Antigen Specific Tregs and related compositions, methods and systems

ABSTRACT

Antigen specific regulatory T cells are described and related compositions, methods and systems. Methods to generate an antigen specific anti-inflammatory regulatory T cell is provided, the method comprising contacting either a T cell or an antigen presenting cell with a zwitterionic polysaccharide conjugated to the antigen for a time and under condition to generate an antigen specific regulatory T cell that is capable of inhibiting a pro-inflammatory response against the antigen.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. application No. 61/346,837,filed on May 20, 2010, which is incorporated by reference herein in itsentirety.

STATEMENT OF GOVERNMENT GRANT

The U.S. Government has certain rights in this invention pursuant toGrant No. DK078938 awarded by the National Institutes of Health.

FIELD OF THE INVENTION

The present disclosure relates to the immune system, and, in particular,to antigen specific regulatory T cells and related compositions, methodsand systems.

BACKGROUND OF THE INVENTION

Regulatory T cells (Tregs) belong to a group of white blood cells knownas lymphocytes, and play a central role in cell-mediated immunity. Inparticular, regulatory T helper cells (also known as suppressor T cellsor Th cells) are a sub-group of lymphocytes (a type of white blood cellor leukocyte) that plays an important role in establishing andmaximizing the capabilities of the immune system and in particular inactivating and directing other immune cells.

In particular, Tregs are a component of the immune system thatsuppresses biological activities of other cells associated to an immuneresponse. More particularly, Tregs can secrete immunosuppressivecytokines TGF-beta and Interleukin 10, and are known to be able to limitor suppress inflammation.

T cells and in particular Tregs are involved in antigen specific immuneresponse. In particular, antigen specific Tregs are functional toregulate and resolve an inflammatory response triggered by antigenspecific inflammatory T cells for clearance of antigens. Antigenspecificity of the inflammatory and regulatory response is paramount toavoid immune-compromising the host.

SUMMARY

Provided herein are antigen specific regulatory T cells and relatedcompositions, methods and systems that in several embodiments are ableto inhibit an antigen specific pro-inflammatory cell mediated and/orhumoral immune response in vitro and/or in vivo.

According to a first aspect, a method to generate an antigen specificanti-inflammatory regulatory T cell is described. The method comprisescontacting a T cell with a zwitterionic polysaccharide conjugated to theantigen for a time and under condition to generate an antigen specificregulatory T cell capable of inhibiting a pro-inflammatory responseagainst the antigen.

According to a second aspect, a method to generate an antigen specificantiinflammatory regulatory T cell is described. The method comprisescontacting a T cell with an engineered Bacteroides fragilis hereindescribed for a time and under condition to generate an antigen specificregulatory T cell capable of inhibiting a inflammatory response againstthe antigen.

According to a third aspect a method to generate an antigen specificanti-inflammatory regulatory T cell is described. The method comprisescontacting an antigen presenting cell with a zwitterionic polysaccharideconjugated to the antigen for a time and under condition to generate anantigen presenting cell presenting the antigen. The method furthercomprises contacting the antigen presenting cell presenting the antigenwith a T cell for a time and under condition to generate an antigenspecific regulatory T cell capable of inhibiting an inflammatoryresponse against the antigen.

According to fourth aspect, a method to generate an antigen specificanti-inflammatory regulatory T cell is described. The method comprisescontacting an antigen presenting cell with an engineered Bacteroidesfragilis herein described for a time and under condition to generate anantigen presenting cell presenting the antigen. The method furthercomprises contacting the antigen presenting cell presenting the antigenwith a regulatory T cell to generate an antigen specific regulatory Tcell capable of inhibiting an inflammatory response against the antigen.

According to a fifth aspect, an engineered Bacteroides fragilis isdescribed, wherein the engineered Bacteroides fragilis expresses aheterologous antigen conjugated with polysaccharide A.

According to a sixth aspect, an antigen specific anti-inflammatoryregulatory T cell is described that is obtainable by a method togenerate an antigen specific anti-inflammatory regulatory T cell hereindescribed.

According to a seventh aspect, a system to generate antigen specific,anti-inflammatory regulatory T cells is described. The system comprisesat least two selected from the group consisting of an engineeredBacteroides fragilis expressing an antigen, a zwitterionicpolysaccharide, a T cell, and an antigen.

According to an eight aspect, a method of inhibiting antigen specificinflammation in an individual is described. The method comprisestreating the individual with a zwitterionic polysaccharide conjugated toan antigen for a time and under conditions to induce an antigen specificregulatory T cell in the individual specific for the antigen.

According to a ninth aspect, a method of inhibiting antigen specificinflammation in an individual is described, the method comprisestreating the individual with an engineered Bacteroides fragilisexpressing an antigen conjugated to polysaccharide A for a time andunder condition to generate an antigen specific regulatory T cellcapable of inhibiting a pro-inflammatory response against the antigen.

According to a tenth aspect, a method of treating a condition of anindividual associated with an antigen specific pro-inflammatory T-cellresponse in the individual is described. The method comprises treatingthe individual with an antigen specific anti-inflammatory T cell,wherein the antigen specific anti-inflammatory T cell is specific forthe specific antigen of the antigen specific pro-inflammatory T cellresponse.

The Tregs herein described and related compositions methods and systemscan be used in connection with medical, pharmaceutical, veterinaryapplications as well as fundamental biological studies and variousapplications, identifiable by a skilled person upon reading of thepresent disclosure, wherein generating antigen specific Tregs isdesirable.

In one embodiment, a method to generate an antigen specificanti-inflammatory regulatory T cell is provided, the method comprisingcontacting either: a) a T cell with a zwitterionic polysaccharideconjugated to the antigen for a time and under condition to generate anantigen specific regulatory T cell that is capable of inhibiting apro-inflammatory response against the antigen; or b) an antigenpresenting cell with a zwitterionic polysaccharide conjugated to theantigen for a time and under condition to generate an antigen specificregulatory T cell that is capable of inhibiting a pro-inflammatoryresponse against the antigen.

In another embodiment, a method to generate an antigen specificanti-inflammatory regulatory T cell is provided, the method comprisingcontacting either: a) a T cell with a zwitterionic polysaccharide for atime and under condition to generate an antigen specific regulatory Tcell that is capable of inhibiting a pro-inflammatory response againstthe antigen; or b) an antigen presenting cell with a zwitterionicpolysaccharide for a time and under condition to generate an antigenspecific regulatory T cell

The details of one or more embodiments of the disclosure are set forthin the accompanying drawings and the description below. Other features,objects, an advantages will be apparent from the description anddrawings, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the detailed description andexample sections, serve to explain the principles and implementations ofthe disclosure.

FIG. 1 shows that PSA functionally expands suppressive regulatory Tcells. FIG. 1A shows flow cytometry (FC) analysis results of thepercentage of Foxp3+ cells within the CD4+CD25+ population in the MLNsof Balb/c mice treated with PSA or PBS every other day for 6 days priorto the rectal instillation of TNBS. These data are representative ofthree independent experiments. FIG. 1B shows FC analysis results of micethat were treated as in FIG. 1A, MLN cells were counted and absolutenumbers of CD4+CD25+Foxp3+ cells determined (×10³). Numbers representthe average of four mice in a single experiment. FIG. 1C shows Foxp3expression in MLNs. RNA was extracted from the MLNs of mice treated asin FIG. 1A and Foxp3 expression normalized to β-actin expression in thetotal lymph node. FIG. 1D shows analysis of CD4+CD25+ cells purifiedfrom the MLNs of colitic PBS or PSA treated mice and incubated with CFSEpulsed CD4+CD25− in an in vitro suppression assay. Numbers indicate thepercentage of cells undergoing at least one cellular division at 2different ratios of effector T cells (Teff) and regulatory T cells(Treg). These data are representative of two independent experiments.FIG. 1E shows FC analysis of Foxp3+ T cells from C57B1/6 mice that wereorally fed PSA every other day for 2 weeks. MLNs were extracted and thepercentage of Foxp3+ T cells within the CD4+CD25+ compartment wasanalyzed by FC. Each symbol represents an individual mouse. The p valuewas determined by a two-tailed students t test. FIG. 1F shows theresults of an in vitro suppression assay of CD4+CD25+ cells that werepurified from the MLN of PBS or PSA treated mice and incubated with CFSEpulsed CD4+CD25− cells, mitomycin C treated CD4− cells and purifiedα-CD3. Numbers indicate the percentage of proliferating cells at 3ratios of Teff to Treg cells. FIG. 1G shows that PSA induces expansionof Tregs, but not B cells, during experimental colitis. The MLN of TNBSmice were analyzed for the presence of B cells by FACS.

FIG. 2 shows that purified PSA activates ‘inducible’ Foxp3+ Tregs. InFIG. 2A, C57B1/6 Foxp3−GFP mice were orally treated with purified PSAevery other day for 6 days. The MLNs were extracted and the CD4+Foxp3+or the CD4+Foxp3−T cells were purified by cell sorting based on ±GFPexpression. RNA was extracted from these cell types and used forqRT-PCR. These data are representative of three independent experiments.FIG. 2B shows that purified PSA treatment upregulates Foxp3 expressionduring TNBS colitis.

FIG. 3 shows that B. fragilis mono-colonization elicits tolerant T cellresponses in the intestinal environment. FIG. 3A shows RT-PCR resultsshowing that mono-association of germ-free animals with B. fragilisincreases the total amount of IL-10 within the colon. RNA was extractedfrom the colon of indicated mice and IL-10 was assayed by qRT-PCR. Eachsymbol represents an individual mouse. Statistical significant wasdetermined by a two-tailed student's t test. FIG. 3B shows results ofC57B1/6 germ-free mice irradiated and reconstituted with bone marrowfrom Foxp3−GFP mice. Mice were subsequently colonized with B. fragilisstrains (as indicated) for 8-10 weeks. Lamina propria lymphocytes werepurified from the colon and re-stimulated with PMA/ionomycin in thepresence of brefeldin A. Each symbol indicates an individual mouse.These data are representative of two independent experiments. FIG. 3Cshows FC analysis of cells extracted and treated as in FIG. 3B. Thepercentage from individual mice of CD4+Foxp3+IL10+ cells in the coloniclamina propria was determined by FC. FIG. 3D shows analysis of genesknown to be associated with regulatory T cells. CD4+Foxp3−GFP+ T cellswere purified from the MLNs by cell sorting. RNA was extracted and usedfor qRT-PCR to analyze genes. FIG. 3E shows DNA extracted from fecalsamples of mice that were irradiated and reconstituted with bone marrowto ensure sterility. Universal 16s primers demonstrate that germfreemice remained sterile throughout the experiment.

FIG. 4 shows that intestinal tolerance toward B. fragilis colonizationis lost in the absence of PSA. FIG. 4A shows FC analysis of laminapropria lymphocytes (LPL) extracted from the colons of indicated animals(n=4/group) and re-stimulated with PMA and ionomycin in the presence ofbrefeldin A for 5 hours. Cells were stained with α-CD4 and α-IL-17A andanalyzed by FC. These data are representative of four independentexperiments. FIG. 4B shows ELISA of IL-17A in colonic LPLs re-stimulatedwith PMA and ionomycin for 24 hours. The relative expression of IL-17A(FIG. 4C) and RORγT (FIG. 4D) from qRT-PCR is shown. CD4+Foxp3− GFP−cells were cell sorted and RNA was extracted and qRT-PCR performed. FIG.4E shows ELISA of CD4+ T cells purified from the MLNs of indicated miceand incubated with plate bound a-CD3 alone (white boxes) or in additionto TGF-β (light grey boxes) or with TGF-β and IL-6 (dark grey boxes).Cells were incubated for 72 hours and IL-17A was determined by ELISA.These data are representative of three independent experiments. FIG. 4Fshows ELISA analysis of intestinal cells from B. fragilisΔPSA colonizedanimals producing increased levels of IL-17 specifically to moleculesfrom B. fragilis. T cell-depleted splenocytes were incubated for 24hours with the indicated strains of heat-killed bacteria. Before use,APCs were washed to remove un-internalized bacteria. Colonic laminapropria lymphocytes from mice mono-associated with either B. fragilis(white boxes) or B. fragilisΔPSA (grey boxes) were incubated withbacterial-pulsed APCs for 72 hours. The amount of secreted IL-17A fromthese cultures was analyzed by ELISA. These data are representative ofthree independent experiments.

FIG. 5 shows that Bacteroides fragilis induces regulatory T celldevelopment. FIGS. 5A and 5B show FACS analysis of CD4+Foxp3−(GFP−)cells. CD4+Foxp3−(GFP−) cells were FACS sorted fromconventionally-colonized animals and equal numbers of cells weretransferred into GF Rag−/−recipients that were either left germ-free, orcolonized by B. fragilis or B. fragilisΔPSA. MLNs were analyzed forCD4+GFP+(Foxp3) T cells. Cells in FIG. 5B were re-stimulated in vitroand stained for a-CD4 and a-IL-10. Cells are gated on CD4+ T cells. ** pvalue of <0.01. FIG. 5C demonstrates that Tregs isolated from the MLNsof B. fragilis colonized animals induce TGF-β in a PSA dependent manner.

FIG. 6 shows that Bacteroides fragilis colonization does not affect thepresence of naturally occurring Tregs but in the absence of PSAinflammatory responses are induced. FIG. 6A and FIG. 6B shows FCanalysis of CD4+Foxp3+ T cells. Mice were colonized as indicated and thepercentage of CD4+Foxp3+ T cells was determined by flow cytometry. Eachsymbol represents an individual mouse. There are no significantdifferences between any groups as determined by a student's t test. Eachsymbol represents an individual animal in FIG. 6B. FIG. 6C shows thepercentage of CD4+IL-17A+ cells within the lamina propria. Thepercentage of CD4+IL-17A+ cells within the lamina propria is representedfrom individual mice from the same experiment. These data arerepresentative of four independent experiments.

FIG. 7 shows that PSA promotes Tregs with suppressive activity in a TLR2dependent manner. FIG. 7A-7B shows percentage of CD4+Foxp3+ cells fromTLR2−/− mice. TLR2−/− mice were fed PSA. MLNs were extracted andanalyzed for the percentage of CD4+Foxp3+ cells. Each symbol representsthe percentage of CD4+Foxp3+ from an individual mouse. NS, notsignificant. FIG. 7B shows qRT-PCR analysis of CD4+CD25hi+ and CD4+CD25−T cell populations that were FACS sorted from MLNs of PSA-fedTLR2−/mice. IL-10 levels were analyzed by qRT-PCR. Light and dark barsindicate IL-10 levels in PBS or PSA treated animals, respectively.Results are representative of two independent trials. FIG. 7C shows thatRORγT expression is elevated in the colons of mice mono-associated withB. fragilisΔPSA. Colons were homogenized and RNA extracted from thetissue. Expression levels of RORγT were assayed by q-PCR.

FIG. 8 shows purified PSA is sufficient to induce functional Foxp3+Tregs with an inducible phenotype. FIG. 8A shows results of an in vitrosuppression assay of CD4+CD25+ Tregs. Mice were fed PBS or PSA andCD4+CD25+ Tregs were purified from indicated animals and titrated intothe assay. Each bar represents one round of division. * indicatesstatistical significance p<0.05. FIG. 8B shows quantitative RT-PCRresults of MLNs. Foxp3−GFP animals were gavaged with purified PSA. MLNswere extracted and CD4+ Foxp3−(GFP−) and CD4+Foxp3+ (GFP+) cellpopulations were sorted and RNA collected. Dark bars representtranscript levels from mice treated with PSA. Lighter bars representdata acquired from animals treated with PBS. Quantitative real time PCRwas performed to determine the levels of ICOS, perforin and Tbet in eachsample. FIG. 8C shows level of expression of T-bet by q-PCR. CD4+Foxp3+cells were purified from the indicated mice and the level of expressionof T-bet analyzed. Cells from the MLNs (FIG. 8D) or splenocytes (FIG.8E) from indicated animals were re-stimulated for 5 hours with PMA andlonomycin in the presence of Brefeldin A. Cells were subsequentlystained for indicated cytokines. Plots are gated on live CD4+ cells.FIG. 8F shows dysregulation of luminal ATP is not the mechanism by whichelevated levels of colonic IL-17 are induced in B. fragilisΔPSA animals.

FIG. 9 shows that Th17 cell responses are induced in the absence of PSA.FIG. 9A shows IL-17A by ELISA in CD4+ T cells. CD4+ T cells werepurified from the MLNs of indicated mice (colonized with differentbacteria as illustrated in the figure) and incubated with plate bounda-CD3 alone (white boxes) or in addition to TGF-β (light grey boxes) orwith TGF-b and IL-6 (dark grey boxes). Error bars represent SD oftriplicate samples, and are representative of three independentexperiments.

FIG. 10 shows that adaptive immune responses to B. fragilis areantigen-specific. FIG. 10A shows secretion of IL-17A by ELISA in colonicLPLS. Colonic LPLs were isolated from either B. fragilis or B.fragilisΔPSA colonized animals and incubated with APCs pulsed with thebacterial species indicated. Error bars represent SD of triplicatesamples. These data are representative of three independent trials. FIG.10B shows IL-17A and IL-10 production. Germ-free animals were irradiatedand reconstituted with bone marrow from Foxp3−GFP animals. Animals weresubsequently left germ-free or colonized with B. fragilis or B.fragilisΔPSA. CD4+Foxp3−(GFP−) cells were purified by FACS from theseanimal groups and transferred into Rag−/−recipients mono-associated withB. fragilisΔPSA. 10 days post-transfer, cells were assayed for IL-17Aand IL-10 production. These data are representative of two independentexperiments. FIG. 10C shows ELISA analysis of B. fragilis-specific IgA.Each symbol represents an individual mouse. Samples were normalized tothe weight of the colonic content collected. * p value of <0.05. FIG.10D shows ELISA analysis of IgA reactivity toward either B. fragilis, B.thetaiotaomicron, or B. vulgatus antigens. Error bars represent SD oftriplicate samples.

FIG. 10E shows total cell extracts from indicated bacteria separated ona polyacrylamide gel and transferred to PVDF membrane. Blots were probedwith antibody preparations from either B. fragilis or B. fragilisΔPSAcolonized animals, and immune-reactive species were detected byanti-mouse IgA secondary linked to HRP. White arrows indicate antigenicbands that are reactive with IgA isolated from B. fragilisΔPSA colonizedanimals but not from wild-type B. fragilis. These data arerepresentative of two independent experiments.

FIG. 11 provides further evidence that adaptive immune responses to B.fragilis are antigen specific. FIG. 11 graphically depicts the resultsof an ELISA of the total amount of IgA in the colonic contents. Valueswere normalized to the weight of the colonic contents isolated. Eachsymbol represents IgA within the colon of a single mouse.

FIG. 12 shows PSA-mediated tolerance to B. fragilis but not otherbacteria. FIG. 12A shows ELISA analysis of the levels of B. fragilis andB. vulgatus reactive IgA. Germ-free mice were co-colonized with equalnumbers of B. vulgatus and either wt B. fragilis or B. fragilisΔPSA andlevels of B. fragilis and B. vulgatus reactive IgA were analyzed byELISA. Data are represented as bacterial specific IgA binding normalizedto weight of colonic content. Error bars are SD from individual mice(n=4). Data are representative of two independent experiments. FIG. 12Bshow FC analysis of IgA reactivity to B. fragilis or B. vulgates. FIG.12C shows B. fragilis-specific IgA (left panel) or total IgA (rightpanel). Germ-free mice were co-colonized with wild-type B. fragilis, B.fragilisΔPSA, or both B. fragilis and B. fragilisΔPSA. ** p value of<0.01. NS, not significant. Data are representative of three independentexperiments. FIG. 12D and FIG. 13F show FC analysis of IgA reactivity toB. fragilis following co-colonization between wild-type B. fragilis andB. fragilisΔPSA. FIG. 12E shows a titration curve (IgA dilutions of 1:6,1:4, and 1:2 from left to right). Error bars in FIG. 12E represent theSD within groups (n=4). Data are representative of three independenttrials.

FIG. 13 shows the PSA-mediated antigen specific tolerance to B. fragilisduring complex intestinal colonization. FIG. 13A shows the colonizationof mice. Feces were collected from animals that were co-associated withwt B. fragilis and B. fragilisΔPSA. Wild-type B. fragilis carried aplasmid that conferred chloramphenicol resistance. Both strainscolonized mice equally. FIG. 13B shows colony forming units of eachbacterial strain tested. Feces were collected from animals that wereco-colonized with wild-type B. fragilis and B. vulgatus and colonyforming units of each bacterial strain was determined by plating. FIG.13C shows reactivity to B. vulgatus antigens using ELISA. Solublecolonic contents were taken from either germ-free, B. fragilis or B.vulgatus mono-associated mice and tested for reactivity to B. vulgatusantigens.

FIG. 14 shows that Foxp3+ Tregs are required for suppression of adaptiveinflammatory responses during mutualism. FIG. 14A shows FC plots of MLNsfrom animals showing ablation of CD4+Foxp3+ Tregs by DT treatment. FIG.14B and FIG. 14C show IL-17A and IFNγ production by CD4+ T cells.Rag-deficient or irradiated germ-free mice were reconstituted with bonemarrow from Foxp3−DTR animals, and colonized with either B. fragilis orB. fragilisΔPSA. Mice were treated with diphtheria toxin (DT) and LP orMLNs were assayed for IL-17A and IFNγ production by CD4+ T cells.Numbers indicate percentage of cells within each quadrant. These dataare representative of three independent experiments. FIG. 14D showsqRT-PCR analysis of expression of IL17A. Colonic LPLs from indicatedmice were isolated and re-stimulated for 24 hours with plate-bound α-CD3and α-CD28. FIG. 14E shows B. fragilis-specific reactivity by ELISA. IgAwas collected from the ileum and colon of indicated animals, and B.fragilis-specific reactivity was determined using a standard ELISAprotocol. These data are representative of three independentexperiments.

FIG. 15 shows further evidence that Foxp3+ Tregs are required forsuppression of adaptive immunity to B. fragilis. FIG. 15A depicts thepercentage of CD4+IFNγ+ T cells in DT treated animals mono-associatedwith either B. fragilis or B. fragilisΔPSA. As showing in FIG. 15B, IgAis not significantly different in the absence of Tregs. Intestinalcontents were extracted as described in materials and methods and totalIgA measured by ELISA. In FIG. 15C PSA specific IgA was measured byELISA and relative units normalized to weight of colonic contents.

FIG. 16 shows that outer membrane vesicles from Bacteroides fragiliscontain PSA. FIG. 16A shows OMVs produced by wild-type B. fragilis andB. fragilisΔPSA that were detected by transmission electron microscopyof EDL (electron dense layer)-enriched B. fragilis. FIG. 16B shows animmunoblot analysis of whole cell (WC) and outer membrane vesicles (OMV)extracts from wild-type and PSA-mutant bacteria. FIG. 17C showsimmunogold labeling of purified OMVs, stained with anti-PSA andanti-IgG-colloidal gold conjugate (5nm), analyzed by electronmicroscopy.

FIG. 17. PSA elicits IL-10 production through TLR2 signaling directly ona T cell. FIG. 17A shows results of experiments where splenic cdllc+cells were enriched from the spleens of WT or TLR2−/− animals andco-cultured with CD4+ T cells purified from the spleens of either WT orTLR2−/− animals along with anti-CD3 and TGF-β in the presence or absenceof PSA for a total of 5 days. Supernatants were collected from culturesand IL-10 was assayed by ELISA. Cultures were performed in triplicate. *indicates p values less than 0.05 and *** indicates p values less than0.005. These results are representative of three independentexperiments. FIGS. 17B-17C show results of experiments where Bone marrowderived dendritic cells (BMDCs) from either IL-10−GFP (as WT control) orTLR2−/− animals were cocultured with CD4+ T cells purified from thespleens of IL-10−GFP (as WT control) or TLR2−/− animals cultured withanti-CD3 and TGF-β in the absence (−) or presence of purified PSA. Thepercentage of CD4 cells expressing GFP (IL-10) is shown. *** indicates ap valued less than 0.01. FIG. 17C shows results of experiments wherecells were cultured as in FIG. 17A and the amount of secreted IFN-γ wasassayed by ELISA. * indicates p values less than 0.05. NS indicates notsignificant.

FIG. 18. PSA can directly signal through TLR2 on a T cell in the absenceof APC and this does not require TLR 1 or TLR6. FIGS. 18A-18B showresults of experiments where CD4+ T cells were isolated from wild-type(WT) TLR1−/−, TLR2−/−, TLR6−/−, or CD 14−/− animals. Cells werestimulated with anti-CD3 in the presence of TGF-β with (+) or without(−) PSA. in vitro cultures were allowed to incubate for 4 days and IL-10secreted into the supernatant was measure by ELISA. * indicatesstatistical significance p=<0.05. Cell cultures were 94% pure. FIG. 18Cshow results of experiments where BMDCs were made from WT, TLR2−/−,animals and incubated with purified CD4+T cells from WT mice. Cultureswere stimulated with anti-CD3 and TGF-β and incubated for 4 days.Secreted IFN-γ were assayed from these cultures by ELISA. * indicatesstatistical significance p=<0.05:

FIG. 19: PSA is a unique TLR2 ligand. FIG. 19A show results ofexperiments performed with PSA, PAM3CysK and FSL1 on CD4+Foxp3−T cells.PAM3CysK is a known TLR1/TLR2 ligand while FSL1 is a known TLR2/6ligand. PSA's ability to induce IL-10 from a highly purified population(greater than 99% pure CD4+Foxp3−T cells) of non-Treg cells was assayed.Non-Treg cells (CD4+Foxp3−) were FACs sorted from Foxp3−GFP animals andstimulated with anti-CD3 in the presence of TGF-β and indicated TLRligands PSA or PAM3CysK. Secretion of IL-10 was assayed by ELISA.

FIG. 20: PSA can enhance Treg function in vitro. FIG. 20 show results ofexperiments where CD4+Foxp3+T cells were FACS sorted from Foxp3−GFP miceand the ability of PSA to enhance the suppressive capacity of a Treg wasmeasured. Purified CD4+CD25− cells were CFSE pulsed and incubated withTregs cells in the presence of BMDCs and anti-CD3 Cell proliferation wasassayed by dilution of CFSE.

FIG. 21: PSA can directly trigger a Treg to produce IL-10 and TGF-B.FIG. 21 shows results of experiments where Facs sorted CD4+Foxp3+ Tcells from WT or TLR2−/− animals were stimulated with anti-CD3 in thepresence of TGF-β and incubated with and without PSA. After 5 days ofculture, RNA was extracted from cells and IL10 was assayed by qRT-PCR.

DETAILED DESCRIPTION

Provided herein are methods and systems for generating antigen specificregulatory T cells.

The term “T cell” as used herein indicates a type of white blood cell orleukocyte including different cell types identifiable by a skilledperson and that include various subtypes of T helper cells or Th cells.The term “regulatory T cells” or “T reg(s)” indicates suppressor Thcells, i.e. Th cells that suppress activation of the immune system andthereby maintain immune system homeostasis and tolerance toself-antigens. In particular a T regulatory cell can be functionallydefined as a CD4+ T cell type that is capable of suppressing immuneresponses such as T helper cell proliferation. Biomarkers that can beused for detection and/or identification of a Treg comprise Foxp3,IL-10, IL-35, CTLA-4, GITR, CD25, TGF, perforin, granzyme B in anycombination. In general, Tregs are crucial for the maintenance ofimmunological tolerance. Their major role in individuals, such ashumans, is to inhibit T cell-mediated immunity toward the end of animmune reaction and to suppress auto-reactive T cells that escaped theprocess of negative selection in the thymus. Two major classes of CD4+regulatory T cells have been described, including the naturallyoccurring Treg cells and the adaptive Treg cells. Naturally occurringTregs are Tregs that develop based on ‘signals’ from the host ororganism that produces the cells (i.e., individuals such as mice andhumans). Adaptive or inducible Tregs are derived from naïve T cells thatreceive signals from the environment (such as gut bacteria for Tregs).

The term “antigen specific regulatory T cell” as used herein indicates aTreg that is capable of suppressing activation of an immune response fora specific antigen, thus inducing tolerance for the specific antigen. Inparticular, an antigen specific Treg is typically a Treg that is capableof suppressing the T helper cell proliferation and inflammation that isspecific for the antigen.

The term “antigen” as used herein indicates a molecule recognized by theimmune system. Exemplary antigens comprise molecules that bindspecifically to an antibody, and any molecule or molecular fragment thatcan be bound by a major histocompatibility complex (MHC) and presentedto a T-cell receptor. “Self” antigens are usually tolerated by theimmune system; whereas “Non-self” antigens are identified as intrudersand attacked by the immune system. Biomarkers that can be used toidentify and/or detect antigen specific Treg typically comprise surfaceexpression of CD25, GITR, CTLA-4, or nuclear expression of Foxp3. Such‘antigens’ can be those associated with various conditions as found inparagraphs [0096] to [0108] below.

The terms “inhibiting”, “inhibit” or “suppressing”, as used hereinindicate the activity of decreasing the biological reaction or process.Accordingly, a substance “inhibits” a certain biological reaction orprocess if it is capable of decreasing that biological reaction orprocess by interfering with said reaction or process. For example, asubstance can inhibit a certain biological reaction or process byreducing or suppressing the activity of another substance (e.g. anenzyme) associated to the biological reaction or process, e.g. bybinding, (in some cases specifically), said other substance. Inhibitionof the biological reaction or process can be detected by detection of ananalyte associated with the biological reaction or process. The term“detect” or “detection” as used herein indicates the determination ofthe existence, presence or fact of an analyte or related signal in alimited portion of space, including but not limited to a sample, areaction mixture, a molecular complex and a substrate. A detection is“quantitative” when it refers, relates to, or involves the measurementof quantity or amount of the analyte or related signal (also referred asquantitation), which includes but is not limited to any analysisdesigned to determine the amounts or proportions of the analyte orrelated signal. A detection is “qualitative” when it refers, relates to,or involves identification of a quality or kind of the analyte orrelated signal in terms of relative abundance to another analyte orrelated signal, which is not quantified.

In one embodiment, “inhibiting”, “inhibit” or “suppressing” can mean anyvalue or amount of the reaction, activity or substance being measuredthat is lower than values found in controls, as determined by those ofskill in the art. For example, a control sample where the reaction isdevoid of PSA or the OMV vesicle and/or the antigen; or a control withonly control media (i.e. PBS control). Similarly, an “increase” or“increasing” activity or amount is any value or amount of the reaction,activity or substance being measured that is greater than values foundin controls, as determined by those of skill in the art.

The terms “inflammatory response”, “pro-inflammatory response” and“inflammation” as used herein indicate the complex biological responseof vascular tissues of an individual to harmful stimuli, such aspathogens, damaged cells, or irritants, and includes secretion ofcytokines and more particularly of pro-inflammatory cytokine, i.e.cytokines which are produced predominantly by activated immune cellssuch as microglia and are involved in the amplification of inflammatoryreactions. Exemplary pro-inflammatory cytokines include but are notlimited to IL-1, IL-6, TNF-a, IL-17, IL21, IL23, and TGF-β. Exemplaryinflammations include acute inflammation and chronic inflammation. Thewording “acute inflammation” as used herein indicates a short-termprocess characterized by the classic signs of inflammation (swelling,redness, pain, heat, and loss of function) due to the infiltration ofthe tissues by plasma and leukocytes. An acute inflammation typicallyoccurs as long as the injurious stimulus is present and ceases once thestimulus has been removed, broken down, or walled off by scarring(fibrosis). The wording “chronic inflammation” as used herein indicatesa condition characterized by concurrent active inflammation, tissuedestruction, and attempts at repair. Chronic inflammation is notcharacterized by the classic signs of acute inflammation listed above.Instead, chronically inflamed tissue is characterized by theinfiltration of mononuclear immune cells (monocytes, macrophages,lymphocytes, and plasma cells), tissue destruction, and attempts athealing, which include angiogenesis and fibrosis. An inflammation can beinhibited in the sense of the present disclosure by affecting and inparticular inhibiting anyone of the events that form the complexbiological response associated with an inflammation in an individual.

An “antigen specific inflammatory response” indicates an inflammatoryresponse specific to a particular antigen, which typically involvesactivation of antigen specific inflammatory T cell or effector Th cellsthat secrete cytokines, proteins or peptides that stimulate or interactwith other leukocytes, including Th cells. Exemplary inflammatory Tcells comprise Th1, Th2 and Th17.

As will be expanded in other parts of this application, any antigen ofinterest can be located or expressed within or on the same vesicle asthe polysaccharide (or PSA), and the vesicle then used to contact witheither a T cell and/or antigen presenting cell, thereby resulting in thegeneration of antigen specific Tregs capable of inhibiting an antigenspecific inflammatory response.

In another embodiment, any antigen of interest can be conjugated topolysaccharides (or PSA) while the polysaccharides are actually locatedon the vesicle (i.e. cell surface conjugation), and then the vesicleused to contact with either a T cell and/or antigen presenting cell;thereby resulting in the generation of antigen specific Tregs capable ofinhibiting an antigen specific inflammatory response.

In yet another embodiment, any antigen of interest can be conjugated topurified natural or synthesized polysaccharides (or PSA) (i.e. notwithin or part of the vesicle), and then the conjugatedpolysaccharide-antigen complex used to contact with either a T celland/or antigen presenting cell; thereby resulting in the generation ofantigen specific Tregs capable of inhibiting an antigen specificinflammatory response.

In one embodiment, T cells, T reg or antigen presenting cells can bepurified from an individual or patient, and then these patient specificcells used to contact with a zwitterionic polysaccharide conjugated tothe antigen for a time in vitro and under condition to generate anantigen specific regulatory T cell that is capable of inhibiting apro-inflammatory response against the antigen.

In another embodiment, the above mentioned generated patient specificantigen specific regulatory T cell of paragraph [0055] can be injectedback into the same individual/patient, who is suffering from a conditioni.e. an inflammatory condition or who has received a graft.

In an embodiment, an antigen specific anti-inflammatory regulatory Tcell is generated by contacting an antigen presenting cell (APC) with azwitterionic polysaccharide conjugated with the antigen for a time andunder condition to generate an APC presenting the antigen and contactingthe APC presenting the antigen with a regulatory T cell for a time andunder condition to generate an antigen specific regulatory T cellcapable of inhibiting anti inflammatory response against the antigen.

The term “contacting” or “incubating” as used herein indicates actionsdirected to creation of a spatial relationship between two itemsprovided for a time and under condition such that at least one of thereciprocal or non reciprocal action or influence between the two itemscan be exerted. In particular, incubation can be performed between aconjugated antigen and a cell and can result in a direct contact and/orinteraction between the antigen and the cell or can result in amodification of the cell following an indirect action of the conjugatedantigen (e.g. following activation or modification of another compoundwhich directly interacts with the cell).

Incubation can also be performed between a first cell and a second cellfollowing contacting of the first cell with an antigen and can result ina direct contact and/or interaction between the first cell and thesecond cell or can result in a modification of the second cell followingan indirect action of the first cell (e.g. following secretion ofcytokines or other molecules which directly interact with the secondcell).

The term “antigen presenting cell” or “APC” as used herein indicates acell that displays an antigen complex with major histocompatibilitycomplex (MHC) on its surface. In particular, antigen presenting cellcomprise as dendritic cell, macrophage, B cells, epithelial cells,fibroblasts, glial cells and additional cells identifiable by a skilledperson.

The term “zwitterionic polysaccharide” or “ZP” as used herein indicatessynthetic or natural polymers comprising one or more monosaccharidesjoined together by glicosidic bonds, and including at least onepositively charged moiety and at least one negatively charged moiety.Zwitterionic polysaccharides include but are not limited to polymers ofany length, from a mono-or di-saccharide polymer to polymers includinghundreds or thousands of monosaccharides. In some embodiments, azwitterionic polysaccharide can include repeating units wherein eachrepeating unit includes from two to ten monosaccharides, a positivelycharged moiety (e.g. an free positively charged amino moiety) and anegatively charged moiety (such as sulfonate, sulfate, phosphate andphosphonate). In some embodiment ZPs can have a molecular weightcomprised between 500 Da and 2,000,000 Da. In some embodiments, the ZPscan have a molecular weight comprised between 200 and 2500. ExemplaryZPs include but are not limited to PSA and PSB from Bacteroidesfragilis, CP5/CD8 from Staphylococcus aureus, and Sp1/CP1 fromStreptococcus pneumonia. Zwitterionic polysaccharides can be isolatedfrom natural sources, and in particular from bacterial sources, e.g. bypurification. Zwitterionic polysaccharides can also be produced bychemical or biochemical methods, as well as by recombinant microorganismtechnologies all identifiable by a skilled person. Thus, those methodsand technologies will not be further described herein in detail.

The wording “polysaccharide A” as used herein indicates a moleculeproduced by the PSA locus of Bacteroides fragilis and derivativesthereof which include but are not limited to polymers of the repeatingunit {→3) α-d-AAT Galp(1→4)-[β-d-Galf(1→3)]α-d-GalpNAc(1→3)-[4,6-pyruvate]-β-d-Galp(1→}, where AATGa1 isacetamido-amino-2,4,6-trideoxygalactose, and the galactopyranosylresidue is modified by a pyruvate substituent spanning O-4 and O-6. Theterm “derivative” as used herein with reference to a firstpolysaccharide (e.g., PSA), indicates a second polysaccharide that isstructurally related to the first polysaccharide and is derivable fromthe first polysaccharide by a modification that introduces a featurethat is not present in the first polysaccharide while retainingfunctional properties of the first polysaccharide. Accordingly, aderivative polysaccharide of PSA, usually differs from the originalpolysaccharide by modification of the repeating units or of thesaccharidic component of one or more of the repeating units that mightor might not be associated with an additional function not present inthe original polysaccharide. A derivative polysaccharide of PSA retainshowever one or more functional activities that are herein described inconnection with PSA in association with the anti-inflammatory activityof PSA.

The terms “conjugated” and “conjugate” as used herein indicates aconnection between two or more compounds and/or substances that allowinternalization of the compounds and/or substances within a sameendosome of an antigen presenting cell, when the conjugatedcompounds/substances are contacted with the antigen presenting cell.Results illustrated in details in the Examples section support theApplicants' conclusion that the co-inclusion of PSA with B. fragilisspecific molecules in outer Membrane Vesicles (OMV) and in particularthe proximity of said molecules with PSA are determinant for the antigenspecific activation of Tregs (see in particular Examples 7-9 and moreparticularly Example 9 wherein PSA produced by B. fragilis fails tosuppress antigen specific response to other antigens present in thegut). As a consequence, the Applicants conclude that the co-inclusion ina same closed environment or direct link between the antigen and a ZP(or PSA) can trigger antigen specific activation of Treg in the sense ofthe present disclosure. Additionally knowledge concerning APCs supportthe conclusion that the connection and proximity is such that aninclusion within a same endosome is allowed when the conjugated antigenand ZP are contacted with an APC.

Accordingly, conjugation in the sense of the present disclosurecomprises physical connection, including direct or indirect linkage andin particular covalent linkage and/or linkage by other chemical bondsbetween the conjugated compounds/substances. Conjugation in the sense ofthe present disclosure also comprises a connection established byinclusion of conjugated compounds/substances within a same/common/sharedvesicle or other enclosed space, and other interactions or relationshipsuch as a spatial relationship consequent to an elevated concentrationof the two or more compounds/substances that allow proximity of theconjugated items in a limited portion of space.

In an embodiment, the zwitterionic polysaccharide can be PSA and/or PSB,as exemplified in the examples section. In an embodiment, PSA or otherZP can be conjugated to the antigen by a physical connection with theantigen such as inclusion of the antigen within a same cellularcompartment where PSA is expressed (see e.g. B. fragilis expressing PSAin mono-associated animals of Examples 7-9). Additional connections thatensure a similar proximity of the ZP with the antigen are expected toprovide similar results are intended to be included within the presentdisclosure and include direct or indirect covalent linkage to theantigen where, for example, ZP is linked to the antigen through a thirdcompound.

In an embodiment, ZP conjugation with the antigen can be performed byinclusion of ZP and the antigen in vesicles formed by a lipid membraneenclosing an aqueous environment. In particular the vesicle can compriseZP and the antigen within the aqueous environment or associated to themembrane. In particular, in an embodiment the vesicle can be formed byB. fragilis outer membrane vesicle (OMV) described in Example 12.

In an embodiment, PSA or other ZP can be conjugated to non-selfantigens, such as those that do not activate a naturally occurring Tregresponse, and can comprise antigens derived from pathogens (e.g.pathogenic bacteria or viruses) or known antigens involved in autoimmunedisease (i.e. MOG peptide). In an embodiment, PSA or other ZP can beconjugated with self antigens that are involved in autoimmune disease.Such autoimmune diseases include, but are not limited to, rheumatoidarthritis; myocarditis; Scleroderma. Type I diabetes, multiplesclerosis, Crohn's disease, ulcerative colitis. Sjorgens syndrome,Hashimoto's thyroiditis, Graves Disease, autoimmune hepatitis, andmyasthenia gravis.

In some embodiments, the effective amount of ZP conjugated to theantigen and in particular PSA and/or PSB is from 25 μg to 100 μg for a25 gram mouse. The results illustrated in the Examples section refer toa dosage of 5 μg/25 gram mouse. Ranges of lower than 10 μg/mouse toabove 200 μgs/mouse are also expected to provide a Treg activation inthe sense of the present disclosure.

In another embodiment, the effective amount of ZPs, antigens, ZP-antigencomplexes, T cells, T regs, antigen presenting cells, and/or vesicles,and any combination thereof, can be determined by those of skill in theart so that a pro-inflammatory immune response is either prevented,inhibited or reduced compared to controls; for instance compared toreactions in the absence of the ZP. Such controls can be designed bythose of skill in the art.

In an embodiment, contacting a conjugated PSA-antigen with the APC canbe performed in absence of Treg and the resulting APC presenting theantigen is subsequently contacted with the Treg. In an embodiment,contacting conjugated PSA-antigen with the APC can be performed inpresence of a Treg and delivered by an antigen presenting cell to theTreg. In those embodiments, conjugation can in particular be performedby inclusion of the antigen and the ZP in vesicles or similarly enclosedspace. In an embodiment, an antigen specific antiinflammatory regulatoryT cell is generated by contacting a T cell with a zwitterionicpolysaccharide conjugated to the antigen for a time and under conditionto generate an antigen specific regulatory T cell capable of inhibitinga pro-inflammatory response against the antigen.

In embodiments, the contacting can be performed by directly incubating aZP conjugated with the antigen with a T cell, and in particular a Tregto activate a tolerogenic immune response. In some of those embodiment,the conjugation can be performed by physical connection and inparticular covalent direct or indirect linkage of the ZP with theantigen. Reference is made to the Treg activation performed by directcontacting of PSA illustrated in the Examples section and in particularin Examples 13 to 19.

Exemplary contacting a ZP conjugated antigen with an antigen presentingcell and/or T cell comprise bathing in vitro a whole sample comprisingone or more types of cells, in a solution containing the antigen undersuitable conditions which depend on the specific cells and the specificantigen and are identifiable by a skilled person upon reading of thepresent disclosure. Additionally exemplary contacting between a ZPconjugated antigen and an antigen presenting cell and/or T cell can beperformed in vitro by introducing the ZP conjugated antigen to a cellculture of purified cells under suitable conditions, and in vivo bytreating an individual with the ZP conjugated antigen.

Additional examples of contacting ZP conjugated antigen with an APC invitro are illustrated in Examples 8 and 9 and FIG. 17 where antigenpresenting cells are purified and incubated with the antigen conjugatedpolysaccharide and subsequently incubated with T lymphocytes to generatea Treg response. In other approaches, an individual can administered theZP conjugated antigen to generate the Tregs in vivo. In particular, theindividual can be treated with a zwitterionic polysaccharide conjugatedto an antigen for a time and under conditions to induce an antigenspecific anti-inflammatory regulatory T cell in the individual specificfor the antigen.

In an embodiment, Treg cells can subsequently be purified out of theindividual. An exemplary antigen specific Treg is show in Example 3 andFIG. 5A. An additional example is provided by FIG. 2A that shows thephenotype of Tregs that are induced in response to PSA treatment andinclude the expression of IL-10, Foxp3, perforin, TGF-B, and granzyme B.

In an embodiment, a T cell with a zwitterionic polysaccharide conjugatedto the antigen for a time and under condition to generate an antigenspecific regulatory T cell capable of inhibiting a pro-inflammatoryresponse against the antigen

Detection of generated Tregs can be performed by using suitable labelsand related suitable techniques identifiable by a skilled person uponreading of the present disclosure

The terms “label” and “labeled molecule” or as used herein as acomponent of a complex or molecule referring to a molecule capable ofdetection, including but not limited to radioactive isotopes,fluorophores, chemiluminescent dyes, chromophores, enzymes, enzymessubstrates, enzyme cofactors, enzyme inhibitors, dyes, metal ions,nanoparticles, metal sols, ligands (such as biotin, avidin, streptavidinor haptens) and the like. The term “fluorophore” refers to a substanceor a portion thereof which is capable of exhibiting fluorescence in adetectable image. As a consequence, the wording “signal” or “labelingsignal” as used herein indicates the signal emitted from the label thatallows detection of the label, including but not limited toradioactivity, fluorescence, chemiluminescence, production of a compoundin outcome of an enzymatic reaction and the like.

Exemplary methods for detection of a biomarker expression can beperformed by methods known to a skilled person including but not limitedto ELISA, Q-PCR and intracellular cytokine staining detected by FACs. Insome embodiments, expression of a biomarker can be detected viafluorescent based readouts on a cell culture performed using an antibodyspecific for the biomarker or molecule associated thereto, labeled withfluorophore, which includes, but not exhaustively, small molecular dyes,protein chromophores, quantum dots, and gold nanoparticles. In anembodiment expression of a biomarker can be detected by detectingexpression of a label under the transcriptional control of a biomarkerpromoter in vivo (e.g., in an animal tissue) or in vitro (e.g. in a cellculture). In some of those embodiments the biomarker can be inparticular IL-10 or Foxp3. An additional method can comprise the use ofa fluorophore called GFP. GFP or green fluorescent protein is able to bedetected by a flow cytometer. GFP is placed under the control of thepromoter that drives expression of either IL-10 or Foxp3. Induction ofeither of these genes by GFP can be used to detect generated Tregs (seee.g. Examples 2 and 3).

In an embodiment, the conjugation can be performed engineering B.fragilis to express the given antigen. Purified conjugatedpolysaccharide will then be incubated with and antigen presenting cells(APC) such as a dendritic cell and then incubated with T regulatorycells to elicit an antigen specific tolerant response.

Inflammatory responses to a particular antigen entity including bacteriathat can be inhibited with the methods and systems herein describedcomprise induction of inflammatory TH17 cells or humoral responses suchas IgA production as shown in Examples 7 to 9 and FIGS. 4, 9, and 10.

In particular, an exemplary antigen specific pro-inflammatory responseherein described comprises induction of TH17 cells such as those shownin FIG. 4. Other inflammatory mediators that can be suppressed by PSA orother ZP include TNF-alpha and IL-1. T effector cell proliferation canalso be suppressed by antigen specific Tregs.

In an embodiment, the contacting can be performed with an engineeredBacteroides fragilis expressing an antigen for a time and undercondition to generate an antigen specific regulatory T cell capable ofinhibiting a pro-inflammatory response against the antigen.

In particular, in embodiments where an engineered B. fragilis is used,the bacteria will be engineered to express an antigen conjugated to PSA.This bacteria can then be orally delivered to the subject. The bacteriawill then be able to colonize the intestine and deliver both PSA and theantigen to the intestinal immune system whereby Tregs will be generatedto that specific antigen.

In embodiments, wherein an engineered B. fragilis is issued conjugationcan be achieved by expressing the antigen in the same cell compartmentas PSA. Thus, conjugated the antigen to purified PSA ensures that theyare delivered together. Also expression of the antigen on the surface ofthe engineered strain of B. fragilis puts the antigen in the samelocation as PSA (on the surface of the bacteria and in particular on theouter membrane).

In an embodiment, an engineered Bacteroides fragilis, is engineeredBacteroides fragilis expresses a heterologous antigen. As mentionedabove, the antigen will be engineered to be expressed on the surface ofthe bacteria so that it is being expressed in the same location as PSA.B. fragilis can be engineered to express an exogenous antigen by placingthe antigenic sequence under the control of a promoter drivingexpression of B. fragilis genes. In particular genes that are known oridentified to be expressed on the outer membrane of B. fragilis (e.g.protein A or other compound identifiable by a skilled person). Forexample, a well characterized cloning vector for B. fragilis, calledpFD340 can be utilized for such heterologous antigen expression.

In an embodiment, a zwitterionic polysaccharide conjugated with anantigen, an engineered B. fragilis expressing the antigen and/or anantigen specific Treg herein described can be administered in a methodof treating or preventing a condition associated with an inflammation inan individual. The method comprises administering to the individual atherapeutically effective amount of the PSA.

The term “therapeutically effective amount” is an amount that results ina reduction, inhibition or prevention of a pro-inflammatory response inthe individual. The amount of ZP or PSA to achieve this can bedetermined by a person of skill in the art.

The term “individual” as used herein includes a single biologicalorganism wherein inflammation can occur including but not limited toanimals and in particular higher animals and in particular vertebratessuch as mammals and in particular human beings.

The term “condition” as used herein indicates the physical status of thebody of an individual, as a whole or of one or more of its parts.Conditions herein described include but are not limited disorders anddiseases wherein the term “disorder” indicates a condition of the livingindividual that is associated to a functional abnormality of the body orof any of its parts, and the term “disease” indicates a condition of theliving individual that impairs normal functioning of the body or of anyof its parts and is typically manifested by distinguishing signs andsymptoms. Exemplary conditions include but are not limited to injuries,disabilities, disorders (including mental and physical disorders),syndromes, infections, deviant behaviors of the individual and atypicalvariations of structure and functions of the body of an individual orparts thereof. Conditions can also include situations where individualshave or about to receive transplanted tissues/organs or grafts. Suchdiseases or disorders can include, but are not limited to, rheumatoidarthritis; myocarditis; Scleroderma; Type I diabetes; multiplesclerosis; Crohn's disease; ulcerative colitis; Sjorgenssyndrome;Hashimoto's thyroiditis; Graves Disease; autoimmune hepatitis;and myasthenia gravis.

The wording “associated to” as used herein with reference to two itemsindicates a relation between the two items such that the occurrence of afirst item is accompanied by the occurrence of the second item, whichincludes but is not limited to a cause-effect relation andsign/symptoms-disease relation.

Conditions associated with an inflammation include but are not limitedto inflammatory bowel disease, including but not limited to Crohn'sdisease and ulcerative colitis, asthma, dermatitis, arthritis,myasthenia gravis, Grave's disease, sclerosis, psoriasis.

The term “treatment” as used herein indicates any activity that is partof a medical care for or deals with a condition medically or surgically.

The term “prevention” as used herein indicates any activity, whichreduces the burden of mortality or morbidity from a condition in anindividual. This takes place at primary, secondary and tertiaryprevention levels, wherein: a) primary prevention avoids the developmentof a disease; b) secondary prevention activities are aimed at earlydisease treatment, thereby increasing opportunities for interventions toprevent progression of the disease and emergence of symptoms; and c)tertiary prevention reduces the negative impact of an alreadyestablished disease by restoring function and reducing disease-relatedcomplications.

In particular, in an embodiment PSA or other ZP can be conjugated with agiven antigen and then administered (e.g. orally or systemically) to theindividual to generate an antigen specific immune response. Thisapproach requires only purified polysaccharide and purified antigen anddoes not necessarily require the generation of the Tregs in vitro butwould instead induce these Tregs in the individual.

In another embodiment, B. fragilis can be engineered as described toexpress the antigen conjugated with PSA and then administered to thesubject. B. fragilis would then be able to colonize the subject andprovide a constant source of PSA and antigen. If B. fragilis is unableto colonize the subject B. fragilis expressing the given antigen couldbe orally administered over a period of time that is expected to becomparable to the time of administration of purified PSA or other ZP. Inanother embodiment, therapeutic effective amounts of ZP-conjugatedantigen comprise dosages that enables a concentration of ZP-conjugatedantigen in the target tissue comprised within the ranges indicatedabove.

In an embodiment, the condition can be graft rejection and the methodwherein engineering Tregs that specifically suppress cellular responsesto donor antigens is expected to increase the success rate of graftacceptance.

In an embodiment, the condition can be rheumatoid arthritis. Depletionof Tregs in mouse models of rheumatoid arthritis increase severity ofdisease, while transfer of Tregs ameliorates disease, indicating thatTregs can play a therapeutic role in the prevention or treatment ofrheumatoid arthritis. Since this disease targets antigens found in thejoints, Tregs can be designed toward joint proteins. Such antigensassociated with rheumatoid arthritis include, but are not limited to,collagen, human chondrocyte glycoprotein 39, proteoglycans, heat shockproteins, citrullinated filaggrin, glucose-6-phosphate isomerase, p205,and BiP.

In an embodiment, the condition can be myocarditis. This disease ismarked by inflammation of the heart muscle. This can result frominfection, exposure to toxic substances or immunologic etiologies thatcan lead to chest pain, heart failure and ultimately death. It has beendemonstrated that mice receiving cells depleted of Tregs develop severemyocarditis, which resembles giant cell myocarditis in humans,indicating that Tregs can be an important factor in preventing this typeof inflammation. Tregs designed to suppress inflammatory responsestoward heart and or heart muscle antigens is expected to be an suitabletherapy for this treating this disease.

In an embodiment, the condition can be Scleroderma. This autoimmunedisease is a chronic inflammatory disease characterized by hardening ofthe skin or other organs. Current treatment includes the use of generalimmuno-suppressants such as methotrexate. The design of Tregs specificfor the skin antigens is expected to be an appropriate treatment. Suchantigens associated with Scleroderma include, but are not limited to,centrosome or centromere autoantigens such as CENP-C, Ufd2, SSSCA1,PM-Scl, and B23.

In an embodiment, the condition can be Type I diabetes. There is a largebody of evidence to support a role for Tregs in controlling inflammationwithin the pancreatic beta cell and thus is expected to be an suitablefor Treg therapy. Such antigens associated with Type I diabetes include,but are not limited to, insulin, proinsulin, chromogranin, and GAD65.

In an embodiment, the condition can be multiple sclerosis. The targetantigens in this disease are well characterized and antigen specificTregs according to the present disclosure. Such antigens associated withmultiple sclerosis include, but are not limited to, Myelin basicprotein, proteosome, B-crystallin, myelin oligodendrocyte glycoprotein,proteolipid protein, Transketolase, enolase, and arrestin.

In an embodiment, the condition can be Crohn's disease or ulcerativecolitis. Our previous data has already demonstrated that PSA cansuppress inflammation within the colon, or current data suggest that PSAinduces Tregs during protection from colitis, thereby making PSA alikely candidate for treatment of intestinal inflammation.

In an embodiment, the condition can be autoimmune diseases such as,Sjorgens syndrome, and the corresponding target antigens can be, but notlimited to, Myelin basic protein, proteosome, B-crystallin, myelinoligodendrocyte glycoprotein, proteolipid protein, Transketolase,enolase, and arrestin.

In an embodiment, the condition can be autoimmune diseases such as SLE(lupus), and the corresponding target antigens can be, but not limitedto, double-stranded DNA, U-1 small nuclear ribonucleoprotein complex.

In an embodiment, the condition can be autoimmune diseases such asHashimoto's thyroiditis and the corresponding target antigens can be,but not limited to, hyroperoxidase (TPO), thyroglobulin (Tg), and thethyroid stimulating hormone (TSH) receptor.

In an embodiment, the condition can be autoimmune diseases such asGraves Disease and the corresponding target antigens can be, but notlimited to, the thyrotropin receptor.

In an embodiment, the condition can be autoimmune diseases such asautoimmune hepatitis and the corresponding target antigens can be, butnot limited to, the 210-kD glycoprotein of the nuclear membrane (GP210), Nucleoporin p62, cyclin A, lamin B receptor' promyelocyticleukemia-associated protein PML, SP100, and CYP 2D6.

In an embodiment, the condition can be autoimmune diseases such asmyasthenia gravis and the corresponding target antigens can be, but notlimited to, the nicotinic acetylcholine receptor.

The ZP, Bacteroides fragilis and/or antigen specific Tregs can beadministered purified conjugated polysaccharide can be delivered eithersystemically (either intraperitoneally or intravenously) or administeredorally. B. fragilis can be delivered orally. In particular,administration is performed with methods and formulation that ensurethat the ZP and antigen are delivered to the target tissues conjugatedone with the other. Accordingly, in some embodiments oral administrationrequires formulations that allow neutralization of the acid in thestomach (e.g. with suitable capsule formulations).

In an embodiments, the engineered Bacteroides fragilis expressing anantigen, the zwitterionic polysaccharide, and the antigen hereindescribed can be provided in a system possibly together with otherreagents suitable to be used in the methods herein described

The systems can be provided in the form of kits of parts. In a kit ofparts, the Bacteroides fragilis, zwitterionic polysaccharide, antigenand other the reagents can be included in one or more compositions, andeach Bacteroides fragilis, zwitterionic polysaccharide, antigen andreagent can be in a composition together with a suitable vehicle.

Additional components can include labels, labeled molecules and inparticular, labeled capture agents specific for an anti-inflammatory oran inflammatory biomarker or a molecule associated to the expressionthereof, a microfluidic chip, reference standards, and additionalcomponents identifiable by a skilled person upon reading of the presentdisclosure.

The term “capture agent” as used herein indicates a compound that canspecifically bind to a target. The wording “specific” “specifically” or“specificity” as used herein with reference to the binding of a firstmolecule to second molecule refers to the recognition, contact andformation of a stable complex between the first molecule and the secondmolecule, together with substantially less to no recognition, contactand formation of a stable complex between each of he first molecule andthe second molecule with other molecules that may be present. Exemplaryspecific bindings are antibody-antigen interaction, cellularreceptor-ligand interactions, polynucleotide hybridization, enzymesubstrate interactions etc. By “stable complex” is meant a complex thatis detectable and does not require any arbitrary level of stability,although greater stability is generally preferred. In some embodiments,the kit can comprise labeled polynucleotides or labeled antibodies.

The components of the kit can be provided, with suitable instructionsand other necessary reagents, in order to perform the methods heredescribed. The kit will normally contain the compositions in separatecontainers. Instructions, for example written or audio instructions, onpaper or electronic support such as tapes or CD-ROMs, for carrying outthe assay, will usually be included in the kit. The kit can alsocontain, depending on the particular method used, other packagedreagents and materials (i.e. wash buffers and the like).

The term “compound” as used herein indicates any chemical substancecomprised of one or more chemical elements and comprises varioussubstances, molecules or component that include but are not limited tobiomolecules and in particular drugs. The term “biomolecule” as usedherein indicates a substance compound or component associated to abiological activity including but not limited to sugars, aminoacids,peptides proteins, oligonucleotides, polynucleotides, polypeptides,organic molecules, haptens, epitopes, biological cells, parts ofbiological cells, vitamins, hormones and the like. The term “drug” asused herein indicates substance that, when absorbed into the body of aliving organism, alters normal bodily function. In particular, drugs inthe sense of the present disclosure include a chemical substance used inthe treatment, cure, prevention, or diagnosis of disease or used tootherwise enhance physical or mental well-being.

The term “mucous membrane” in the sense of the present disclosureindicates a lining of mostly endodermal origin, covered in epithelium,which are involved in absorption and secretion. In an individual, mucousmembranes line various body cavities that are exposed to the externalenvironment and internal organs. Mucous membranes are at several placescontinuous with skin: at the nostrils, the mouth, the lips, the eyelids,the ears, the genital area, and the anus.

A “vesicle” in the sense of the present disclosure is a supramolecularcomplex formed by a membrane forming lipid and additional moleculesassembled in an aqueous environment. In particular, in vesicles hereindescribed the membrane forming lipids are arranged in a lipid layerenclosing an internal aqueous environment herein also indicated ascytosol.

The term “membrane forming lipid” or “amphipatic lipid” as used hereinindicates a lipid possessing both hydrophilic and hydrophobic propertiesthat in an aqueous environment assemble in a lipid layer structure thatconsists of either one or two opposing layers of amphipatic moleculesknown as polar lipid. Each polar lipid has a hydrophilic moiety, i.e., apolar group such as, a derivatized phosphate or a saccharide group, anda hydrophobic moiety, i.e., a long hydrocarbon chain. Exemplary polarlipids include phospholipids, sphingolipids, glycolipids, ether lipids,sterols and alkylphosphocholins. Amphipatic lipids include but are notlimited to membrane lipids, i.e. amphipatic lipids that are constituentsof a biological membrane, such as phospholipids likedimyrisoylphosphatidylcholine (DMPC) or Dioleoylphosphoethanolamine(DOPE) or dioleoylphosphatidylcholine (DOPC). In an embodiment, themembrane of the vesicle is formed by a lipid bilayer mimicking a plasmamembrane (a biological membrane separating the interior of a cell fromthe outside environment, and enclose an aqueous environment) and inparticular the outer membrane of B. fragilis.

Vesicles herein described also comprise a lipopolysaccharide (LPS)either associated with the membrane of the vesicle or comprised in theaqueous environment of the vesicle. The term “lipopolysaccharide” asused herein indicates large molecules consisting of a lipid and apolysaccharide joined by a covalent bond; they are found in the outermembrane of Gram-negative bacteria, act as endotoxins and elicit strongimmune responses in animals. In particular, vesicles herein describedcomprise one or more LPS of B. fragilis which are identifiable by askilled person.

Vesicles herein described can also comprise a peptidoglycan eitherassociated with the membrane of the vesicle or comprised in the aqueousenvironment of the vesicle. The term “peptidoglycan” as used hereinindicates a polymer consisting of sugars and amino acids that forms amesh-like layer outside the plasma membrane of bacteria (Eubacteria, notArchaebacteria), forming the cell wall. In particular, vesicles hereindescribed comprise one or more peptidoglycans of B. fragilis which areidentifiable by a skilled person.

In one embodiment the vesicle comprising the zwitterionic polysaccharide(i.e. PSA) or PSA itself can be termed a “compound” or part of a“composition” to be administered to an individual or patient in need oftreatment. For instance, in patients who have inflammation orinflammatory disorders.

Additional compounds that can be comprised in the vesicles, comprisemembrane proteins, membrane lipids carbohydrates and nucleic acids, andin particular, membrane proteins, membrane lipids carbohydrates andnucleic acids of B. fragilis.

Exemplary vesicles in the sense of the present disclosure comprise smallmembrane-enclosed sacs that can store or transport substances. Vesiclescan form naturally because of the properties of the membrane forminglipid, or they may be prepared from bacterial membranes. Most vesicleshave specialized functions depending on what materials they contain onthe membrane and/or the aqueous environment.

In an embodiment, the vesicles herein described are formed by portionsof membranes of bacteria. In an embodiment, vesicles are formed by OuterMembrane Vesicles (OMVs) of the bacteria.

In some embodiments, where the composition is to be administered to anindividual the composition can be a pharmaceutical composition, andcomprise one or more vesicles each comprising PSA. In a more particularembodiment, the pharmaceutical composition can comprise of one or morevesicles each comprising PSA and one or more of another compound, and/ora pharmaceutically acceptable or appropriate carrier/vehicle.

In another embodiment, the above pharmaceutical composition, comprisingone or more vesicles each comprising PSA and one or more of anothercompound, and/or a pharmaceutically acceptable or appropriatecarrier/vehicle, wherein an individual/subject with an inflammatorycondition or inflammation given this composition shows an improvement.

In some embodiments, the vesicles herein described can be included inpharmaceutical compositions together with an excipient or diluent. Inparticular, in some embodiments, pharmaceutical compositions containvesicles herein described, in combination with one or more compatibleand pharmaceutically acceptable vehicle, and in particular withpharmaceutically acceptable diluents or excipients.

The term “excipient” as used herein indicates an inactive substance usedas a pharmaceutically acceptable or appropriate carrier for the activeingredients of a medication. Suitable excipients for the pharmaceuticalcompositions herein disclosed include any substance that enhances theability of the body of an individual to absorb vesicles hereindescribed. Suitable excipients also include any substance that can beused to bulk up formulations with vesicles herein described to allow forconvenient and accurate dosage. In addition to their use in thesingle-dosage quantity, excipients can be used in the manufacturingprocess to aid in the handling of vesicles herein described. Dependingon the route of administration, and form of medication, differentexcipients may be used. Exemplary excipients include but are not limitedto antiadherents, binders, coatings disintegrants, fillers, flavors(such as sweeteners) and colors, glidants, lubricants, preservatives,sorbents.

Pharmaceutically acceptable or appropriate carriers can be, but notlimited to, organic or inorganic, solid or liquid excipient which issuitable for the selected mode of application such as oral applicationor injection, and administered in the form of a conventionalpharmaceutical preparation. Such preparation includes solid such astablets, granules, powders, capsules, and liquid such as solution,emulsion, suspension and the like. Said carrier includes starch,lactose, glucose, sucrose, dextrine, cellulose, paraffin, fatty acidglyceride, water, alcohol, gum arabic and the like. If necessary,auxiliary, stabilizer, emulsifier, lubricant, binder, pH adjustorcontroller, isotonic agent and other conventional additives may beadded.

The pharmaceutically acceptable or appropriate carrier may well includeother compounds known to be beneficial to an impaired situation of thegut, (e.g., antioxidants, such as Vitamin C, Vitamin E, Selenium orZinc); or a food composition. The food composition can be, but is notlimited to, milk, yoghurt, curd, cheese, fermented milks, milk basedfermented products, ice-creams, fermented cereal based products, milkbased powders, infant formulae, tablets, liquid bacterial suspensions,dried oral supplement, or wet oral supplement.

The term “diluent” as used herein indicates a diluting agent which isissued to dilute or carry an active ingredient of a composition.Suitable diluent include any substance that can decrease the viscosityof a medicinal preparation.

In certain embodiments, compositions, compounds, and, in particular,pharmaceutical compositions can be formulated for enteral administrationincluding, but not limited to, i) by mouth (orally) as tablets,capsules, or drops; ii) by gastric feeding tube, duodenal feeding tube,or gastrostomy; and enteral nutrition; and iii) rectally as asuppository.

In some embodiments, vesicles herein described comprising PSA can beused in a method of treating or preventing a condition in an individual.

The method comprises administering to the individual an effective amountof the composition or pharmaceutical composition. The term “individual”as used herein includes a single biological organism whereininflammation can occur including but not limited to animals and inparticular higher animals and in particular vertebrates such as mammalsand in particular human beings.

Further details concerning the identification of the suitable carrieragent or auxiliary agent of the compositions, and generallymanufacturing and packaging of the kit, can be identified by the personskilled in the art upon reading of the present disclosure.

EXAMPLES

The methods and systems herein described and the related compositionsare further illustrated in the following examples, which are provided byway of illustration and are not intended to be limiting.

In particular the following examples relate to generation of antigenspecific T regulatory cells specific for B. fragilis. In particular,illustrated herein are Tregs generated by PSA and/or B. fragilis thatare able to specifically inhibit immune responses elicited by the hostto B. fragilis itself. A skilled person will appreciate theapplicability of the methods and systems herein exemplified for PSAconjugated to B. fragilis or to B. fragilis expressing PSA to anyzwitterionic polysaccharide conjugated to another antigen, includingother bacteria or pathogens or molecules associated thereto in view ofthe teaching of the present disclosure. Additionally, a skilled personwill appreciate the applicability of the methods and systems hereinexemplified to administration of the antigen for treatment or preventionof immune mediated diseases where a given antigen/s are driving thedisease, antigen specific Tregs can be engineered to suppress theseimmune responses.

The following material and methods were used for all the methods andsystems exemplified herein.

Mice and Bacteria. 8-10 week old SPF (Specific Pathogen Free) C57B1/6mice were purchased from Taconic Farms. 8-10 week old SPF Balb/c micepurchased from Taconic were used for the TNBS model of colitis.Foxp3−GFP on C57B1/6 background were a kind gift from Talal Chatila(University of California, Los Angeles). Foxp3−GFP mice were devoid ofHelicobacter species. TLR 2−/− mice were purchased from Jackson.Germ-free C57B1/6 and Rag−/− mice were bred in plastic Trexler isolatorsat Caltech, fed autoclaved food and water, and screened weekly by PCRand microbiological plating to ensure sterility. To obtain germ-freeC57B1/6 Foxp3−GFP or Foxp3−DTR bone marrow chimeras, C57B1/6 or Rag−/−germ-free mice were lethally irradiated and reconstituted with bonemarrow from Foxp3−GFP donors by retroorbital injection or intraorbitalinjection. Mice were immediately placed in newly autoclaved cages andwater supplemented with antibiotics (100 mg/ml gentamicin and 10 mg/mlerythromycin) throughout the 2 month reconstitution period. Mice werecolonized by oral gavage with strains of B. fragilis NCTC9343 that areresistant to erythromycin and gentamicin. For transfer experiments,germfree Rag−/− mice were sublethally irradiated 24 hours prior to celltransfer. For diphtheria toxin experiments, mice were given 50 μg/kg ofdiphtheria toxin intraperitoneally (i.p) for two consecutive days andevery third day thereafter. Mice were sacrificed between day 10-14 posttreatment. All mice were fed LabDiet 500010 chow, and were cared forunder IACUC guidelines from the California Institute of Technology.

In vitro Suppression Assay. Either CD4+CD25+ or CD4+Foxp3+ cells wereused as a source of Tregs. CD4+CD25− cells were pulsed with 1 ml of a 5mM CFSE stock for 10 minutes at 37° C. CFSE labeled cells were washed inPBS twice and immediately used. 1×10⁵ mitomycin C (Sigma) treated CD4depleted splenocytes were mixed with CFSE-pulsed CD4+CD25− (or Foxp3−)responder cells. Indicated dilutions of CD4+CD25+ Treg cells weretitrated in and 1 μg/ml of anti-CD3 was added in a round bottom 96 wellplate. Cultures were incubated for 3-4 days and then analyzed by flowcytometry.

Quantitative Real-Time Polymerase Chain Reaction. RNA was collected fromindicated cells using Trizol (Invitrogen). cDNA was made using aniSCRIPT cDNA syn in vitro thesis kit per manufacturer's instructions(Bio-Rad). qRT-PCR reactions were performed using IQ SYBR Green Supermixper manufacturer's instructions (Bio-Rad). Reactions were run on theBio-Rad IQ5 q-PCR machine. Primers are as follows ICOS: F 5′-TAC TTC TGCAGC CTG TCC AT3′(SEQ ID NO:1) & R 5′-CAG CAG AGC TGG GAT TCA TA-3′ (SEQID NO:2); FOXP3:F 5′-GCA ATA GTT CCT TCC CAG AGT TCT-3′ (SEQ ID NO:3) &R 5′-GGA TGG CCC ATC GGA TAA G-3′ (SEQ ID NO:4); IL-10: F-5′ CTG GAC AACATA CTG CTA ACC G-3′ (SEQ ID NO:5) & R 5′-GGG CAT CAC TTC TAC CAG GTAA-3′ (SEQ ID NO:6); EBI3:F 5′-AGC AGC AGC CTC CTA GCC T-3′(SEQ ID NO:7)& R 5′-ACG CCT TCC GGA GGG TC-3′(SEQ ID NO:8); GITR: F-5′ TGC CCA GCTATA CCC TTG GT-3′ (SEQ ID NO:9) & R5′ CCG CTC TCA TAC ACC CAC TTC-3′(SEQID NO:10); CD25: F 5′-AAC CATAGT ACC CAG TTG TCG G-3′ (SEQ ID NO:11) & R5′-TCC TAA GCA ACG CAT ATA GAC CA3′ (SEQ ID NO:12); L32: F 5′-AAG CGAAAC TGG CGG AAA C-3′ (SEQ ID NO:13) & R 5′TAA CCG ATG TTG GGC ATCAG-3′(SEQ ID NO:14).

Experimental Colitis. 8 week old Balb/c mice were purchased fromTaconic. Animals were pretreated with 50 μg of PSA every other day for 6days prior to administration of TNBS. 0.75-1.5% TNBS (Sigma) in 50%ethanol was rectally instilled using a 3.5 Fr silicone catheter (Instechsolomon). Mice were weighed daily until necropsy 5 days post-TNBSadministration.

Lamina Propria Lymphocyte Extraction. The colon was carefully cleaned ofthe mesentery and residual fat and cut open longitudinally and then cutinto large fragments (1-1.5 cm). Fragments were placed in 50 ml conicaland rinsed well with ice cold PBS (Invitrogen). Cleaned intestinalfragments were placed in 15 ml of epithelial cell dissociation solution(Ca+ and Mg+ free HBSS with 5 mM EDTA and 10mM Hepes) at 37° C. for 15minutes with gentle agitation (100 rpm). This step was repeated oncemore. The fragments were then minced with a razor blade and then placedin a digestion solution (HBSS with 5% FBS, 3 units/ml of Dispase, 0.5mg/ml of Collagenase D and 0.5 mg/ml of DNAase I (all from WorthingtonBiochemical), digested for 20 minutes with slow rotation at 37° C. andthen vortexed well. Supernatants were collected by filtering through a40 μm cell strainer. Digestions were repeated two more times, LPLsre-suspended in 8 ml 40% Percoll and layered this on top of 5 ml of 80%Percoll (GE Healthcare). LPLs were recovered from the interface of the40 and 80% gradient after centrifugation, washed and used as described.

Intracellular Cell Staining. For Foxp3 intracellular staining, 0.5-1×10⁶cells were first surface stained then permeabilized and fixed in 100 mlof Fixation and Permeabilization buffer (eBiosciences). For IL-10 andIFNγ intracellular cytokine staining, lamina propria lymphocytes wereextracted and re-stimulated with 750 ng/ml of ionomycin and 50 ng/ml ofPMA (Calbiochem) in the presence of 0.5 μl of GolgiPlug (BD biosciences)for 4-5 hrs at 37° C. Cells were subsequently surface stained and fixedwith 2% paraformaldehyde. 1×10⁶ cells were permeabilized overnight with100 μl of Fixation and Permeabilization buffer (eBiosciences). Cellswere stained with 0.3 μg of either anti-IL-17A or IL-10 for 20 minutesat 4° C. All antibodies were purchased from eBiosciences.

Bacterial Antigen Preparation. Bacterial cultures were harvested andwashed extensively with PBS and sonicated. Disrupted cultures were spunat 10,000×g for 20 minutes and the supernatant was collected. Bradfordwas used to determine protein concentration. Supernatants were stored at−20° C. until use.

Bacterial FACS. The protocol of Slack et al. was used to directlymeasure IgA binding to bacteria (Slack et al., 2009). Briefly, 1 ml ofbacterial culture was washed with buffer (PBS 1% BSA, 0.05% sodiumazide). Soluble colonic contents were diluted 1:10 in buffer and furtherdiluted (1:2, 1:4, 1:6). 25 μl of antibody solution and 25 μl ofbacterial suspension were mixed and incubated at 4° C. for 1 hour.Bacteria were washed before staining with a monoclonal PE-amouse IgA(1:250; Ebiosciences) for 30 minutes at 4° C. Bacteria were washed,fixed in PFA and analyzed by flow cytometry using FSC and SSC parametersin logarithmic mode.

IgA immunoblot. 1 ml of bacterial culture was pelleted and washedextensively and resuspended in 1 ml of PBS and sonicated. Bacteriallysates were spun at 10,000×g for 20 minutes and the supernatantsubjected to Bradford. Equal amounts of lysates were run on an SDSpolyacrylamide gel and transferred to PVDF membrane. Membranes wereblocked in 5% milk overnight at room temperature. Equal amounts ofsoluble colonic contents (as measured by Bradford) were used to probethese membranes at 4° C. overnight. Membranes were washed extensivelyand subsequently probed with a biotin conjugated anti-mouse IgA andstreptavidin-HRP.

Colonic IgA Collection and ELISA. The small intestine or colon was openlongitudinally and the intestinal contents (including feces and mucus)were collected in 500 μL of PBS with proteinase inhibitor cocktail(Roche). Samples were weighed and spun at 8000×g for 10 minutes at 4° C.Supernatants were collected and stored at −20° C. until use. Forbacterial specific IgA ELISAs, a 96 well plate was coated with 2 μg/mlof lysates collected from B. fragilis, B. thetaiotaomicron, or B.vulgatus in PBS (100 μL/well) overnight at 4° C. Plates were blockedwith Assay Diluent (eBiosciences) for 1 hour at room temperature. 10 μLof colonic contents were diluted in 100 μL of assay diluent and seriallydiluted 1/10⁴ additional times. Samples were left on overnight at 4° C.Anti-mouse IgA conjugated to biotin was used at 1/1000 dilution for 1hour at room temperature and streptavidin-HRP (Southern Biotech) wasused at 1/1000 dilution for 1 hour at room temperature.

Statistics. Differences between data sets were analyzed by Mann-WhitneyU-test or student's t test using Microsoft excel or Prism 5.0.

Example 1 PSA Induces Development and Expansion of T regs in Presence orAbsence of Inflammation and the Induced Tregs are FunctionallySuppressive In Vitro and In Vivo

The prominent human symbiont Bacteroides fragilis prevents intestinalinflammation and experimental colitis through production of the capsularpolysaccharide, PSA (27). To determine the impact of PSA on Foxp3+ Tregsduring protection from experimental colitis, immune cells were analyzedfrom animals that were induced for intestinal inflammation and orallytreated with purified PSA. TNBS treatment results in a T cell-mediatedcolonic immune response, as treated animals lost a significant amount ofweight, displayed marked thickening of the colon, lymphocyteinfiltration and epithelial hyperplasia (data not shown and Ref (27)).As previously reported, disease was not evident in TNBS-treated animalsthat were fed PSA.

Vehicle treated (PBS) and PBS treated TNBS animals (TNBS+PBS) had asimilar percentage of Treg cells within the mesenteric lymph nodes(MLNs) (FIG. 1A). Consistent with PSA's anti-inflammatory properties,mice fed PSA reproducibly had a 10% increase in the percentage of Foxp3+cells within the CD4+CD25+ compartment of the MLNs (FIG. 1A).

Additionally, the absolute number of CD4+CD25+Foxp3+ cells in the MLNswas significantly higher in PSA-treated animals when compared to PBS orvehicle treated animals (FIG. 1B). PSA expansion of the Foxp3+ Tregpopulation is specific, as the percentage of B cells in the MLNs did notdiffer between PBS and PSA fed mice (FIG. 1G). Consistent with anincrease in the percentage of Foxp3+ cells in PSA treated mice, therewas an increase in the expression of Foxp3 transcripts in total MLNs(FIG. 1C). It was also found that Foxp3 expression was increased on aper cell basis in CD4+CD25+ cells during PSA mediated protection fromcolitis (FIG. 2B), demonstrating that PSA up-regulates proportional andcell-intrinsic Foxp3 expression. It was previously reported that PSAtreatment of animals expands an unknown CD4+ T cell subset (7); thecurrent results suggest that this population may be a CD4+CD25+Foxp3+Treg cell.

One of the primary functions of Treg cells is to suppress the activationand proliferation of inflammatory T effector cells. The functionalcapacity of a Treg cell can be assessed by measuring in vitrosuppression of proliferation by naïve CD4+CD25− T cells pulsed with thefluorophore, CFSE (dilution of this dye is proportional to rounds ofcell division). The suppressive capacity of Tregs during PSA mediatedprotection from experimental colitis was determined by the addition ofvarying amounts of CD4+CD25+ Treg cells purified from the MLNs ofvehicle (PBS only-no TNBS), and PBS-or PSA-treated colitic mice. Asexpected over 90% of the effector cells underwent proliferation in theabsence of Tregs (data not shown), that was partially suppressed whenTregs from vehicle or PBS treated colitic mice were added to the culture(FIG. 1D). Notably however, Tregs isolated from the MLNs of PSA fed mice(TNBS+PSA) suppressed T cell proliferation to a significantly higherdegree than cells from untreated animals (43.5% proliferating cells vs.63.5% at a 1:2 Teff:Treg ratio), demonstrating Tregs from animalsprotected from colitis by PSA have increased functional suppressiveactivity.

Dramatic changes occur during an intestinal inflammatory response;including the expansion of antigen specific T and B lymphocytes,activation of innate immune cells, and secretion of copious amounts ofinflammatory cytokines. These events result in a complex cytokine milieuwith the capacity to influence a countless array of immune pathways.During the steady state however, many of these cytokines are notexpressed and the cellular and molecular intestinal environment is verydifferent than that seen during immunity.

To determine whether PSA expands Tregs during homeostasis (such asduring commensal colonization), mice were fed PSA and theCD4+CD25+Foxp3+ population of Tregs within the MLNs during the steadystate monitored. Mice treated with PSA consistently had an increasedpercentage of Foxp3+ cells within the CD4+CD25+ T cell subset in the MLN(FIG. 1E). And further during homeostasis, Tregs isolated fromPSA-treated animals displayed a greater ability to suppress effector Tcell responses compared to control animals (28.5% proliferating cellsvs. 43.0% at a 1:2 Teff:Treg ratio) (FIG. 1F).

The findings illustrated in the present example demonstrate that PSAinduces functional Tregs in presence or absence of intestinalinflammation.

Example 2 PSA can Induce Development of Functionally Suppressive TregsThrough MHCTCR Recognition

The microbiota has profound influences on the development and functionof the immune system (Macpherson and Harris, 2004). Colonization ofgerm-free animals with Bacteroides fragilis represents a model systemfor the study of immune-bacterial symbiosis (Mazmanian et al., 2005).Since recent studies have shown a critical role for Treg-produced IL10during maintenance of intestinal homeostasis (Rubtsov et al., 2008), itwas necessary to understand how B. fragilis colonization affects Foxp3+Treg development and cytokine production.

Germ-free C57B1/6 mice were mono-associated with wild-type B. fragilisor a strain deleted of PSA (B. fragilisΔPSA) (FIGS. 4, 6). Consistentwith recent studies (Atarashi et al., 2008; Ivanov et al., 2008), thepercentage of CD4+Foxp3+ Tregs within the colon did not differsignificantly between groups (FIG. 6), suggesting that the microbiotadoes not affect naturally occurring Tregs cells within the intestine.

Intriguingly, production of the IL-10 transcript is deficient in theabsence of the microbiota; moreover B. fragilis colonization restoresproduction of IL-10 in the colon in a PSA-dependent manner (FIG. 3A).Mono-association of germ-free animals with B. fragilis results in asignificant increase in the proportion of IL-10-producing CD4+Foxp3+Tregs (FIG. 3). The induction of IL-10 from Foxp3+ T cells by B.fragilis is dependent on PSA, as Tregs from B. fragilisΔPSA colonizedanimals have similar IL-10 levels as germ-free mice. Colonization withB. fragilis directs IL-10 production almost exclusively from Foxp3+ (andnot Foxp3−) T cells (FIGS. 2A, 3D). Compared to conventionally-colonizedanimals, germ-free mice display lower expression levels of IL-10, Foxp3and TGF-β2 produced by CD4+Foxp3+ Tregs (FIG. 2A and FIG. 5C).Remarkably, B. fragilis mono-association restores expression of theseanti-inflammatory genes, a phenotype that is completely PSA dependent.Natural Treg markers such as CD25 are not altered. Thus, PSA induces thedevelopment of Foxp3+IL-10+ Tregs in the gut during normal colonizationof animals.

To examine whether PSA is able to direct de novo Treg conversion,CD4+Foxp3−T cells were purified from Foxp3−GFP animals (Lin et al.,2007) and adoptively transferred into germ-free Rag−/− lymphopenic mice.Groups of animals were either left germ-free or colonized with wild-typeor B. fragilisΔPSA. FIG. 5A shows that while Treg conversion does notoccur in germ-free mice, colonization with wild-type B. fragilis inducedsignificant levels of Foxp3+ Tregs. PSA is required for Treg conversion,as B. fragilisΔPSA colonized animals contained Treg cell numberscomparable to germ-free animals (FIG. 5A). Furthermore, Foxp3+ T cellsin B. fragilis-colonized animals acquired IL-10 expression (FIG. 5B).

Collectively, CD4+Foxp3+IL-10+ Treg lineage differentiation in the colonrequires gut bacteria, revealing PSA as the first bacterial molecule ofthe intestinal microbiota that regulates Foxp3+ Treg development.

Example 3 Tregs Induced by PSA Exhibit a Characteristic and PSA-SpecificAntiinflammatory Gene Expression Profile

Tregs potently restrain inflammatory responses through the secretion ofIL-10, TGF-β and IL-35 (Collison et al., 2007; Maynard et al., 2007).Additionally, contact-dependent mechanisms include effector T cellcytolysis through the secretion of perforin and granzymes (Gondek etal., 2005), as well as expression of anti-inflammatory surface receptorssuch as GITR and CTLA-4 (Vignali et al., 2008).

Many subsets of Tregs exist within the Foxp3+ population; therefore tounderstand how PSA affects the functional capacity of Foxp3+ Tregs, theexpression of Treg-associated genes in response to PSA treatment wasanalyzed. Foxp3−GFP mice (where green fluorescent protein marks Foxp3+cells) were orally treated with PSA (or PBS control), and geneexpression analysis was performed on both CD4+Foxp3+ or CD4+Foxp3−Tcells from the MLNs. As expected, Treg-associated genes including IL-10,TGF-β2, were dramatically increased in Foxp3+ compared to Foxp3− T cells(PBS samples; FIG. 2A). Remarkably, PSA induces over 8-fold increasedlevels of IL-10 from CD4+Foxp3+ Tregs, but had virtually no impact onCD4+Foxp3− T cells.

Accordingly, PSA elicited significant induction of TGF-β2 in Foxp3+ Tregcells. PSA treatment also significantly increases the transcription ofgranzyme B, perforin and CCR6, a chemokine receptor shown to beassociated with the migration of Treg cells (FIG. 2A) (Yamazaki et al.,2008). It is important to note that PSA does not globally impact allTreg derived cytokines as expression of TGF-β1 and Ebi3 is not altered,demonstrating specificity for a distinct Treg profile. Furthermore,production of the ‘natural’ Treg-associated surface molecules CTLA-4,GITR, CD25 and ICOS are not changed among Foxp3+ cells in response toPSA treatment (FIG. 2A). Taken together, these data suggest that PSAactivates ‘inducible’ Foxp3+ iTregs and reveals a PSA-specific geneexpression program within Foxp3+ Treg cells.

In a separate experiment, Foxp3−GFP mice were gavaged with purified PSA(or PBS control), and RNA was extracted from either CD4+Foxp3−non-Tregor CD4+Foxp3+ Treg cells of the MLNs following FACS purification. Asexpected, gene expression in Foxp3− and Foxp3+ T cell subsets differeddramatically and included higher basal levels of IL-10, TGF-β1, CD25,GITR, ICOS, and CTLA-4 in Foxp3+ T cells (FIG. 2A). It was previouslyreported that IL-10 production by an unknown CD4+ T cell population isrequired for PSA-mediated protection from intestinal inflammation (27).Very intriguingly, PSA induces over 8-fold increased levels of IL-10from CD4+Foxp3+ Tregs than that expressed in PBS-treated cells (FIG.2A). While PSA marginally up-regulated TGF-β2 expression in non-Tregcells, it elicited over 7-fold induction of TGF-β2 in Treg cells. PSAtreatment also significantly increases the transcription of granzyme Band perforin from Foxp3+ Tregs. It is important to note that PSA doesnot globally impact all Treg-derived cytokines as expression of Ebi3 andTGF-β1 is not altered, demonstrating specificity for a PSA-induced Tregprofile. Furthermore, production of the ‘natural’ Treg-associatedsurface receptors CD25, GITR, ICOS, and CTLA-4 are not changed amongFoxp3+ cells in response to PSA treatment (FIG. 2A).

Taken together, these data suggest that PSA activates ‘inducible’ Foxp3+Tregs that suppress inflammation through cytokine and cytolyticmechanisms, revealing a PSA-specific gene expression program within Tregcells.

Example 4 T regs Induced by PSA Actively Engender T Cell ToleranceThrough IL10 Production and/or Activation of a Th1 Profile

To determine whether PSA alone is sufficient to expand Tregs,conventionally-colonized mice were fed PSA and proportions of CD4+Foxp3+Tregs within the MLNs were monitored. Mice orally treated with PSAdisplay increased percentages of CD4+Foxp3+ T cells in the MLNs (FIG.1A). One of the primary functions of Treg cells is to suppress theactivation and proliferation of inflammatory T effector cells. Tregfunction was determined by the addition of various ratios of CD4+CD25+Treg cells purified from MLNs to naïve CD4+CD25− T cells. It was foundthat Tregs isolated from PSA-treated animals display considerablygreater ability to suppress in vitro T cell responses compared toPBS-treated control animals (e.g., 28.5% proliferating cells vs. 43.0%at a 1:2 Treg:Teff ratio) (FIG. 1F). These findings demonstrate that PSAinduces functional Foxp3+ Tregs with enhanced suppressive capacity inconventionally-colonized animals.

Applicants previously reported that colonization of germ-free animalswith PSA-producing bacteria increases the Th1 cytokine interferon-g(IFNγ) among splenic CD4+ T cells (Mazmanian et al., 2005). Inparticular, Applicants previously showed that Tregs induced by PSA havethe ability to direct Th1/Th2 response (see Mazmanian, et al 2005). Th1responses are generally believed to be controlled by the transcriptionfactor T-bet; however, a recent report has revealed that a subset ofFoxp3+ Tregs express T-bet, but lack IFNγ expression (Koch et al.,2009).

It was now found that CD4+Foxp3+ T cells within the MLNs of wild-type B.fragilis colonized animals express T-bet in a PSA-dependent manner (FIG.8B). Intriguingly, these cells do not produce IFNγ (FIG. 8D). However,splenic CD4+ T cells from the same animals express IFNγ from non-Tregsin a PSA-dependent manner (FIG. 8). Remarkably, splenic T cells do notproduce IL-10, unlike those of the MLNs.

These results reveal a compartmental difference in the ability of PSAinduced Treg to elicit a protective response. In particular, while PSAinduced Tregs are capable to induce a Th1 profile in the spleen,corresponding Tregs in the gut promote tolerance through production ofIL-10. Additionally, the ability of PSA promoting development ofFoxp3+IL10+ Treg cells in the gut, further supports the notion that PSAactively engenders mucosal tolerance.

Example 5 B. fragilis Mono-Association of Mice Elicits Tolerant a PSADependent Treg Response Specific to B. fragilis

Germ-free mice have numerous developmental and functional defectssuggesting that the microbiota has profound influences on the intestinalimmune response (4). Monoassociation of germ-free mice provides an idealmodel system to analyze the physiological contributions of individualbacterial species, and assign molecular functions through comparativecolonization with bacterial mutants. To further understand how B.fragilis colonization affects Treg cell development, germ-free C57B1/6mice were lethally irradiated (to deplete all hematopoietic cells) andthey were reconstituted with bone marrow from Foxp3−GFP animals. Micewere subsequently left germ-free or mono-associated with wild-type B.fragilis or a strain deleted of PSA (B. fragilisΔPSA).

Mice were monitored weekly by PCR to ensure their microbiological status(FIG. 3E). Consistent with recent reports (21, 23), the percentage ofTregs (CD4+Foxp3+) cells residing within either the MLN or colon did notdiffer significantly between groups (data not shown), suggesting thatthe microbiota is not a requirement for the presence of naturallyoccurring Tregs cells within the intestine. However, IL-10 productionamong Foxp3+ Tregs within the intestine is deficient in the absence ofthe microbiota; moreover B. fragilis colonization restores theproduction of IL-10 within the colon in a PSA dependent manner (FIG.3A). Mono-association of germ-free animals with B. fragilis results inan over 2.5-fold increase in the percentage of IL-10-producing Tregswithin the colon (FIGS. 3B and 3C). The induction of IL-10 from Foxp3+cells by B. fragilis is almost completely reliant on PSA, as Tregsanalyzed in B. fragilisΔPSA colonized animals have similar IL-10 levelsas germ-free mice. Recent studies have shown that IL-10 production fromFoxp3+ Tregs is critical for maintaining homeostasis at mucosal surfaces(31). Thus, commensal colonization by B. fragilis directs thedevelopment of functional IL-10-producing Tregs within the intestinalcompartment

To investigate the impact of microbial colonization on the geneexpression profile of Tregs, CD4+Foxp3+ T cells were purified from theMLN of conventionally colonized, germfree, B. fragilis or B.fragilisΔPSA mono-associated mice. Germ-free mice display a deficiencyin IL-10, Foxp3, and TGF-β2 expression within the CD4+Foxp3+ Tregpopulation when compared to conventionally colonized mice (FIG. 3D). B.fragilis monoassociation completely restores expression of all three ofthese genes. Induction of IL-10, Foxp3, and TGF-β2 is completelydependent on PSA production by B. fragilis, as monoassociation ofanimals with B. fragilisΔPSA does not elevate the expression of thesesuppressive genes. The changes in gene expression are specific toinducible Treg markers as CD25 is not impacted by homeostatic microbialcolonization. Thus, colonization by symbiotic bacteria has a dramaticimpact on the programming of Treg-associated genes, demonstrating thatB. fragilis induces a tolerogenic intestinal immune environment duringhost mutualism.

Example 6 PSA Activity on Treg Requires Toll-Like Receptor 2 Signaling

Many microbial products are sensed by pattern recognition receptors suchas toll-like receptors (TLRs). Though historically believed to induceinflammation, a series of studies now show that TLR signaling can alsopromote anti-inflammatory responses (reviewed in (van Maren et al.,2008)). PSA has recently been shown to coordinate cytokine productionfrom innate immune cells through TLR2 signaling (Wang et al., 2006);however a role for TLR2 in Treg development remains unknown. Tounderstand the mechanism by which PSA coordinates Treg biology,TLR2-deficient animals were treated orally with PSA and analyzed forCD4+Foxp3+ T cell development. In contrast to the Treg expansion seen inwild-type animals (FIG. 7A), no difference in the percentage ofCD4+Foxp3+ T cells in TLR2−/− mice treated with PSA was observed (FIG.7). Additionally, induction of IL-10 by Tregs in response to PSA is lostin the absence of TLR2 expression (FIG. 7B). The findings are entirelyconsistent with reports that TLR2 knockout mice have defects inFoxp3+Treg cells (Liu et al., 2006; Sutmuller et al., 2006). Thoughfurther work is needed to fully understand how innate immune signalingcontributes to Treg lineage differentiation, PSA-mediated Tregdevelopment is a TLR2 dependent mechanism.

Example 7 In Absence of PSA B. fragilis Induces an Inflammatory ImmuneResponses Specific for B. fragilis

Almost all bacteria share microbial ligands for pattern recognitionreceptors (e.g., LPS (endotoxin), peptidoglycan, unmethylated CpG, etc),suggesting that molecular mechanisms must allow the mucosal immunesystem to distinguish between symbiotic and pathogenic bacteria. Currenttheories for this discrimination include spatial separation between theimmune system and the microbiota (immunologic ignorance), as well asinnate immune suppression (Hooper, 2009). To control microbialinfections, the immune system elicits the function of pro-inflammatoryTh17 cells. Tregs control Th17 immunity in order to prevent collateraldamage to host tissues, and Treg function is required for suppression ofimmune reactions to both innocuous non-self and self antigens.Furthermore, symbiotic and pathogenic bacteria share many microbialligands for pattern recognition receptors, suggesting that molecularmechanisms must allow the immune system to discriminate betweenbeneficial and harmful bacteria.

It was reasoned that during its lifelong colonization of the mammalianintestine, B. fragilis (and presumable other symbionts) must betolerated as ‘self’ in order to prevent deleterious inflammation in thehost. Therefore, it was hypothesized that PSA may have evolved to allowimmunologic tolerance to B. fragilis antigens by suppressing Th17responses. Consistent with published literature (21, 23), germ-freeanimals have virtually no Th17 cells within the colon when compared withconventionally colonized animals (FIG. 4A and FIG. 6C) (FIG. 9A). WhileB. fragilis mono-associated animals do not significantly elicit Th17cells within the colon, the absence of PSA results in substantiallyincreased intestinal Th17 cell responses. Colonic lamina proprialymphocytes isolated from B. fragilisΔPSA mono-associated animals haveincreased secretion of IL-17A (FIG. 4A) (FIG. 9) and elevatedtranscription of RORγt, the Th17-specific lineage differentiation factor(FIG. 4D) (FIG. 7C). Additionally, CD4+Foxp3−T cells purified from theMLNs of B. fragilisΔPSA colonized animals have increased IL-17A andRORγt levels (FIG. 4C and FIG. 4D).

No signs of colitis were observed in animals with increased Th17 cellsTh17 cell differentiation occurs in response to T cell receptorstimulation in the presence of TGF-β and IL-6. To determine themagnitude of T cell responses from differentially colonized animals,CD4+ T cells were purified from MLNs and assayed for the capacity ofcells to produce IL-17A during in vitro Th17 skewing assays. Cells fromB. fragilis mono-associated animals have equivalent levels of IL-17Aproduction as germ-free animals, even in the presence of TGF-β and IL-6.Most notably, CD4+ T cells from animals colonized with B. fragilismissing PSA (B. fragilisΔPSA) display significantly increased levels ofIL-17A production compared to cells recovered from wild-type colonizedanimals) (FIG. 4E).

These data indicate that T cells isolated from B. fragilismono-associated animals are intrinsically resistant to Th17differentiation. ATP has recently been demonstrated as a mechanism bywhich the intestinal microbiota can initiate development of Th17 cellswithin the intestine (23). While elevated levels of luminal ATP areconsistently found in conventionally colonized animals, germ-free and B.fragilis mono-associated animals have significantly lower levels of ATPthat is not changed by the absence of PSA, ruling out a role for ATPdysregulation as a cause of the increased IL-17 seen in B. fragilisΔPSAanimals (FIG. 8F). These data reveal that a commensal bacterium of thehuman microbiome can indeed induce an intestinal immune response similarto a pathogen. However, through the dedicated production of PSA,proinflammatory Th17 development against B. fragilis is activelysuppressed, supporting a model by which PSA-mediated tolerance, and notimmunologic ignorance, prevents Th17 responses to B. fragilis.

The data demonstrate that B. fragilis is not simply ignored by the hostimmune system, but rather actively induces a tolerogenic intestinalenvironment through its dedicated production of PSA. It was wondered ifthe host inflammatory response that ensues in the absence of PSArepresents non-specific immune cell priming or is directed toward B.fragilis. To test this notion, pro-inflammatory immune responses byintestinal cells were measured from either B. fragilis or B.fragilisΔPSA mono-associated animals that were challenged withautologous or heterologous commensal bacteria. Isolated colonic laminapropria cells were activated with antigen presenting cells (APCs) pulsedwith various strains of heat-killed commensal bacteria, and IL-17production from thesecultures was analyzed. Minimal IL-17 induction wasdetected in the absence of antigen (no bacteria) (FIG. 4F). Moreover,the addition of antigens provided by incubation of APCs with Bacteroidesthetaiotaomicron or Bacteroides vulgatus elicited only basal levels ofIL-17 by cells isolated from either B. fragilis or B. fragilisΔPSAmono-associated animals.

Remarkably, while lamina propria cells from colons of B. fragilismono-associated animals induced little IL-17 in response to B.fragilis-derived antigens, cells isolated from mice colonized with B.fragilisΔPSA elicit significant amounts of IL-17 following stimulationwith B. fragilis (and not other closely related Bacteroides species).Furthermore, IL-17 production by cells from B. fragilisΔPSA colonizedanimals was lower when APCs were pulsed with wild-type B. fragilis,indicating that PSA has anti-inflammatory properties in vitro, aspreviously reported (27). Enhanced IL-17 production by B. fragilisΔPSAcells is observed in MLNs as well (FIG. 4E). Therefore the findingsreveal that in the absence of PSA, the host mounts an inflammatoryresponse exclusively toward B. fragilis, suggesting that this commensalbacterium evolved PSA to suppress inflammation toward itself duringhost-bacterial mutualism.

Example 8 Th17 Cell Responses to B. fragilis are Antigen-Specific

It was wondered if the host inflammatory response that ensues in theabsence of PSA represents non-specific T cell activation or is directedtoward antigens of B. fragilis. To distinguish between these twopossible mechanisms, IL-17 production by T cells was measured fromeither B. fragilis or B. fragilisΔPSA colonized animals to antigens ofvarious commensal bacteria. Antigen presenting cells (APCs) were pulsedwith bacterial extracts, and co-cultured with lamina propria lymphocytesharvested from animals colonized with B. fragilis or B. fragilisΔPSA. Noadditional stimulation was added, faithfully measuring antigen-specificresponses. Minimal IL-17 induction was detected in the absence ofbacteria (FIG. 9A).

APCs pulsed with Bacteroides thetaiotaomicron or Bacteroides vulgatusantigens elicited only basal levels of IL-17 by cells from both B.fragilis and B. fragilisΔPSA mono-associated animals. Moreover,equivalent IL-17 production between both groups shows that cells from B.fragilisΔPSA colonized mice are not more reactive to non-B. fragilisantigens. Astonishingly, while cells from wild-type B. fragilismono-associated animals induced negligible responses, mice colonizedwith B. fragilisΔPSA elicited significant amounts of IL-17 followingco-culture with B. fragilis-pulsed APCs (but not other closely relatedBacteroides species). Consistent with previous studies, responses arelower when APCs are pulsed with wild-type B. fragilis compared to B.fragilisΔPSA, indicating that PSA has anti-inflammatory activity duringin vitro cell cultures (Mazmanian et al., 2008). Equally enhanced IL-17production from B. fragilisΔPSA animals is observed in cells from MLNsas well (FIG. 9A).These findings reveal that in the absence of PSA, thehost mounts an inflammatory IL-17 response selectively toward B.fragilis antigens.

The data suggest that Th17 cell responses to B. fragilis antigensdevelop in the absence of PSA. To test this concept in vivo, germ-freemice were reconstituted with bone marrow from Foxp3-GFP animals and leftgerm-free, or mono-associated with either B. fragilis or B.fragilisΔPSA. This approach allows Th17 cell induction in the presenceor absence of PSA. CD4+Foxp3-effector T cells were purified from all 3colonized groups and transferred into B. fragilisΔPSA mono-colonizedRag-deficient animals. Th17 cell responses were analyzed as a measure ofT cell reactivity to B. fragilis antigens. Effector T cells derived fromB. fragilis mono-associated animals had minimal expression of Th17 cellswhen transferred to animals that were colonized with B. fragilisΔPSA(FIG. 5A), indicating negligible reactivity toward B. fragilis antigens.However, mice receiving cells from B. fragilisΔPSA colonized donorselicited a significant increase in the percentage of Th17 cells uponre-exposure to B. fragilis antigens. These data show that in vivo Th17cell development to B. fragilis antigens is prevented by PSA.

Example 9 PSA Suppresses Antigen-Specific IgA Responses Against B.fragilis

Mucosal immunoglobulin A (IgA) is produced toward gut bacteria duringcolonization (Slack et al., 2009); therefore it was asked if PSA alsoprevents the development of anti-B. fragilis antibody production. Levelsof B. fragilis-specific IgA were measured in animals mono-associatedwith B. fragilis, B. fragilisΔPSA, or the closely related species B.thetaiotaomicron and B. vulgatus. Intestinal antibody responses werehighly specific as colonic antibody isolated from either germ-free, B.thetaiotaomicron or B. vulgatus mono-colonized animals had no reactivityto B. fragilis antigens (FIG. 4F). Additionally, intestinal IgA from B.fragilis mono-associated animals did not react to antigens from B.thetaiotaomicron or B. vulgatus (FIG. 11D). Consistent with the findingsof increased T cell responses, animals colonized with B. fragilisΔPSAdisplayed elevated levels of B. fragilis-specific IgA contrary to B.fragilis expressing PSA (FIG. 11C).

To determine the nature of the antigens recognized by the host in the B.fragilisΔPSA colonized animals, bacterial extracts were separated on apolyacrylamide gel and probed with IgA from either B. fragilis or B.fragilisΔPSA mono-associated animals. IgA isolated from B. fragiliscolonized animals is species specific, as no reactivity is seen toantigens from B. thetaiotaomicron or Escherichia coli (FIG. 4F). Agreater number and higher intensity of antigenic species were detectedwhen B. fragilis extracts were probed with IgA isolated from B.fragilisΔPSA mono-associated animals compared to wild-type colonizedmice (FIG. 10C). Moreover, total IgA levels are not significantlydifferent between B. fragilis or B. fragilisΔPSA colonized animals (FIG.12), demonstrating specific increases in reactivity to B. fragilisantigens only. Taken together, these data demonstrate that B. fragilisevolved PSA to suppress antigen-specific adaptive immunity duringhost-bacterial mutualism and that the Treg activation triggered by PSAis specific to B. fragilis.

Example 10 PSA Prevents IgA Responses to B. fragilis but not OtherCommensal Bacteria and Actively Engenders Antigen Specific MucosalTolerance

Mono-association studies are informative in determiningantigen-specificity of immune responses. The findings show that PSAsuppresses adaptive immunity to B. fragilis, however they can beinterpreted as: 1) PSA induces tolerance specifically to B. fragilisantigens; 2) PSA induces a tolerant immune environment to antigens ofother bacteria. To distinguish between these two possibilities, animalswere co-colonized with B. fragilis and B. vulgatus, and IgA responseswere measured to both organisms residing in the same microbiota. B.fragilis and B. vulgatus colonize animals to equal levels (FIG. 12).While antibody levels for B. fragilis antigens were again suppressed byPSA during co-colonization, the presence or absence of PSA had no effecton IgA reactivity to B. vulgatus (FIG. 13). Antibody responses arespecific as soluble colonic contents isolated from germfree or B.fragilis mono-associated animals do not react to antigens derived fromB. vulgatus (FIG. 13). Using a quantitative flow-cytometry based assay,it was determined that animals colonized with B. fragilisΔPSA generateremarkably more IgA to B. fragilis surface antigens (FIGS. 12B and 12C).Low levels of binding are likely due to phase variation of surfaceantigens (Liu et al., 2008). No difference in binding to B. vulgatusantigens was observed during co-colonization with either wild-type or B.fragilisΔPSA (FIGS. 12B, 12C and 13), demonstrating that PSA is unableto promote tolerance to antigens of other commensal bacteria residing inthe same microbiota. These results establish that PSA does not induce ageneral, tolerogenic immune environment in the gut during homeostaticcolonization.

Foxp3+ Tregs suppress through dominant tolerance. If PSA induces Tregsthat mediate tolerance to B. fragilis antigens, then wild-type bacteriashould induce Tregs that prevent reactions to antigens of B.fragilisΔPSA. Remarkably, when animals harbor both wild-type and B.fragilisΔPSA in equivalent numbers (FIG. 11), no increases in IgAreactivity to B. fragilis antigens are observed (FIG. 12), showing thatPSA-producing bacteria prevent reactions to B. fragilis cells lackingPSA. The levels of B. fragilis-specific IgA during co-colonization areequivalent to that of wild-type mono-colonized animals, and are specificas total IgA levels are not altered (FIG. 11). Furthermore, IgArecognition to B. fragilis antigens is not elevated duringco-colonization using a quantitative FC assay (FIG. 12E). Based on thisevidence, it is concluded that PSA expressed by B. fragilis mediatesdominant tolerance to its antigens, but not to B. vulgatus antigensfound in the same microbiota. Collectively, the studies confirm that PSAactively engenders antigen-specific mucosal tolerance.

Example 11 Foxp3+ Tregs are Required for Suppression of AdaptiveImmunity to B. fragilis

To verify that Foxp3+ Tregs provide the mechanism for PSA-mediatedsuppression of adaptive immune responses, Th17 and IgA responses to B.fragilis colonization following specificity ablation of CD4+Foxp4+ Tcells was examined. Foxp3-DTR mice express the diphtheria toxin receptor(DTR) under the control of the Foxp3 promoter. Treatment of mice withdiphtheria toxin (DT) results in ablation of Foxp3+ T cells and allowsfor functional analysis of Tregs in vivo (Kim et al., 2007). Consistentwith previous findings (in FIG. 14), Foxp3-DTR reconstituted germ-freeanimals colonized with B. fragilisΔPSA harbor increased Th17 cells inboth the colon (LP) and MLN when compared with animals colonized withwild-type B. fragilis (FIGS. 14A and 14B; left panels, −DT).

This difference is Th17-specific, as there is no effect on IFNgproducing CD4+ T cells from the same tissues (FIG. 14C). Mostimportantly, Treg ablation in B. fragilis mono-associated animalsresults in a dramatic increase in Th17 cells in both the colonic laminapropria and MLN, but does not enhance Th17 responses in B. fragilisΔPSAanimals (FIG. 14B; right panels, +DT). IFNg production in both groups ofmice equally increases in the MLN following DT treatment (FIG. 14B),again showing specificity for Th17 responses. Th17 cell levels actuallydecreased in B. fragilisΔPSA colonized mice. Although this unusualphenotype cannot be explained, the results clearly show specificincreases in Th17 for wild-type bacteria as the percentage of CD4+IL-17+cells increases only in this group following DT administration (FIG.14A). Treatment of mice with diphtheria toxin (+DT) confirms nearlycomplete ablation of Foxp3+ T cells in animals (FIG. 14A). Accordingly,expression of IL-17A transcript in the colonic LP is significantlyincreased in DT-treated animals (FIG. 14D). These data reveal that PSAinduces functional Foxp3+ Tregs that actively suppress Th17 responses toB. fragilis during commensal colonization.

Finally, B. fragilis-specific antibody production in the presence orabsence of Tregs was determined. B. fragilis mono-associated animalsproduce B. fragilis-specific antibodies in both the ileum and colon(FIG. 15). Upon Foxp3+ Treg ablation, B. fragilis-specific IgA increasessignificantly throughout the small and large intestine, with thegreatest increase (over 10-fold) in the colon (FIG. 15). This reflects aquantitative increase in antigen-specific reactivity, as bothPSA-specific and total IgA antibody production throughout the intestineis not affected by the absence of Tregs (FIG. 15). Collectively, it isshown that B. fragilis produces an immunomodulatory molecule thatinduces Treg cells in the gut, which suppress antigen-specific adaptiveimmune responses. These findings reveal a novel cellular and molecularmechanism for how mammals tolerate symbiotic bacterial molecules duringhost-bacterial commensalism.

Example 12 Immunomodulatory Capsular Polysaccharide PSA is ActivelySorted into OMVs of B. fragilis

Ultrathin sections of EDL-enriched B. fragilis were prepared asdescribed in materials and methods and imaged by transmission electronmicroscopy.

The results illustrated in FIG. 16 show that OMVs were abundantlyproduced by bacteria, and could be observed budding from the bacterialenvelope (FIG. 17A, higher magnification). Applicants' previous studieshave shown that deletion of PSA abrogates the immunomodulatory capacityof B. fragilis (Mazmanian, Liu, Tzianabos, and Kasper (2005) AnImmunomodulatory Molecule of Symbiotic Bacteria Directs Maturation ofthe Host Immune System. Cell 122:1 107-118.) (Mazmanian, Round, andKasper (2008) A microbial symbiosis factor prevents intestinalinflammatory disease. Nature. 453 (7195) 620-625. Electron micrographsof a PSA mutant strain (B. fragilisΔPSA) illustrate no defect in OMVsynthesis, and the size, shape and abundance of OMVs produced wereindistinguishable from wild-type bacteria (FIG. 17A). In particular, theresults illustrated in FIG. 17A reveal that vesicles are activelybudding from the surface of bacteria.

To determine if PSA is associated with OMVs of B. fragilis, purifiedvesicles from wild-type and ΔPSA bacteria were subjected to immunoblotanalysis as described in the materials and methods section.

The results illustrated in FIG. 16B show that the vesicles fromwild-type displayed immunoreactivity for PSA, unlike OMVs from B.fragilisΔPSA. B. fragilis produces at least 8 distinct capsularpolysaccharides which coat the surface of bacterial cells, namedPSA-PSH. While PSB was also detected in vesicle preparations, PSG wasabsent, demonstrating selectivity for certain polysaccharides to bepackaged with OMVs (FIG. 17B). Accordingly, the results of FIG. 17B showthat PSA and PSB are associated with vesicles, while PSG is only foundon the bacterial surface. Deletion mutants for capsular polysaccharidesconfirm specificity of each antiserum.

The results from immunoblot analysis were confirmed by experiments ofimmunogold labeling performed as described in the materials and methodssection. The results of immunogold labeling of purified vesiclesillustrated in FIG. 17C and confirm that PSA is physically associatedwith OMVs, and that the vast majority of OMVs from wild-type B. fragilisstain positively for PSA (data not shown). To verify that the absence ofPSA did not alter the molecular composition of OMVs, a proteomicanalysis was performed by mass spectrometry which revealed no majorqualitative or quantitative differences in the protein compositionbetween vesicles from wild-type or PSA-mutant bacteria (data not shown).

PSA is a heterogeneous polymer of repeating subunits. Size separation ofPSA recovered from whole cell extracts by chromatography was performedas well as an immunoblot analysis with anti-PSA of capsularpolysaccharide preparations from whole cells and purified OMVs asindicated in material and methods.

The relevant results illustrated in FIG. 16D surprisingly show that onlythe low molecular weight species is associated with OMVs, illustratingspecificity of PSA packaging into vesicles. In particular, the resultsof FIG. 16D show that only low molecular weight PSA (LPSA) is packagedinto vesicles unlike the high molecular weight (H-PSA) species thatremains associated with the bacterial cell envelope.

Together, the above results reveal that the immunomodulatory capsularpolysaccharide PSA is actively sorted into OMVs of B. fragilis.

Example 13 PSA elicits IL-10 Production Through TLR2 Signaling Directlyon a T Cell

PSA was contacted with splenic cells or Bone marrow derived dendriticcells (BMDCs) co-cultured with CD4+ T cells purified from the spleen, ina series of experiments illustrated in FIGS. 17A to 17D. The resultssupport the conclusion that PSA can elicit IL-10 production through TLR2signaling on a T cell.

Example 14 PSA can Directly Signal Through TLR2 on a T Cell in theAbsence of APC and this does not Require TLR 1 or TLR6

PSA was contacted with T cells isolated from wild-type (WT) TLR1−/−,TLR2−/−, TLR6−/−, or CD14−/− animals in a set of experiments illustratedin FIG. 18A-18B. The results indicate that PSA can directly stimulatethe T cell (in the absence of an APC) to induce IL-10. Induction ofIL-10 by PSA requires TLR2 but does not require TLR1 or TLR6. Since TLR2does not act as a homodimer and is known to heterodimerize with TLR1 andTLR6 this indicates that PSA is acting uniquely through TLR2 to induceIL-10.

PSA was also contacted with BMDCs from WT, TLR1−/−, TLR2−/−, TLR6−/− andCD 14−/− animals and with purified CD4+T cells from WT mice in a set ofexperiments illustrated in FIG. 18. The results indicate that under theexperimental conditions the ability of purified PSA to elicit IL-10production does not depend on signaling through TLR1, TLR2, or TLR6 onthe dendritic cell. However, according to these results IFN-γ productiondoes require both TLR1 and TLR2 signaling on the dendritic cell.

Example 15 PSA is a Unique TLR2 Ligand

PSA, TLR1/TLR2 ligand PAM3CysK and TLR2/6 ligand FSL1 were contactedwith CD4+ Foxp3− T cells in a set of experiments illustrated in FIG. 19.The results indicate that PSA can directly stimulate a non-Treg cell toproduce IL-10. It does so uniquely as other TLR2 ligands like PAM3CysKdo not induce IL-10 production from this same population of non-Tregcells. Additionally, other TLR2 ligands actually suppress IFN-γproduction by the T cell again indicating the unique ability of PSA toactivate a T cell.

TLR 2 ligands, PSA, PAM3CysK, or FSL1 were contacted with CD4+ T cellsin a set of experiments illustrated in FIG. 20. The results indicatethat PSA elicits more IL-10 production from T cells in the absence of anAPC than other known TLR2 ligands.

PSA was contacted with CD4+CD25−T cells from either WT or TLR2−/−animals in a set of experiments illustrated in FIG. 20. The resultsindicate that the induction of IL-10 by non-Treg cells by PSA isdependent on TLR2 signaling on the T cell.

Example 16 PSA can Directly Trigger a Treg to Produce IL-10 and Enhancesthe Survival of Tregs in an In Vitro Culture

CD4+Foxp3+ T cells were stimulated with anti-CD3 in the presence ofTGF-β and incubated with and without PSA in a set of experimentsillustrated in FIG. 21. The results indicate that PSA can directly causea Treg to induce gene expression of IL-10.

CD4+Foxp3+ T cells were incubated with BMDCs and the culture wasstimulated with anti-CD3 and TGF-β in a set of experiments illustratedin FIG. 21. The results indicate that PSA can enhance the survival orproliferation of a Foxp3 expressing T regulatory cell in vitro

The results illustrated in Examples 13 to 16 support the conclusionsthat 1) That PSA acts on TLR2 in the T cell to induce IL-10 but notIFNγ, 2) PSA does NOT require TLR 1 or TLR 6 on the T cell for IL-10production; 3) PSA can directly act on a T cell to induce il-10 (no APCsneeded), Also PSA seems to act differently than other TLR2 ligands (2and 3 are important for highlighting the unique nature of PSA as a tollligand; 4) PSAs ability to induce IL-10 directly on t cell is TLR2dependent; 5) one exp shows PSA can convert to a higher degree in vitro(both % and absolute #); 6) PSA can induce IL-10 from purified Tregs aswell and also seems to maintain/expand a cd4foxp3high population

Bacteroides fragilis, produces a bacterial molecule that can regulatehost immunity to suppress inflammatory responses toward its ownantigens, thereby resulting in host tolerance. Polysaccharide A (PSA) ofB. fragilis induces a specific gene expression profile in functionallysuppressive Foxp3+ regulatory T cells, and coordinates the establishmentof a tolerogenic immune environment. Most notably, host cells recoveredfrom animals colonized with B. fragilisΔPSA are more reactive to B.fragilis, but not to other (closely related) commensal bacteria. Thesedata demonstrate that the host immune system is not ignorant to thepresence of B. fragilis, but rather that PSA is actively suppressingthese inflammatory responses. It appears that commensal bacteria are notimmunologically ignored, and induction of inflammation is a defaultresponse to foreign microorganisms (both commensal and pathogenic). Muchlike virulence factors employed by pathogens, it appears commensalbacteria have evolved symbiosis factors to control host immunity byprogramming the immune system to prevent antigen-specific responsesduring gut colonization. Conversely to pathogens, colonization by B.fragilis actually has beneficial consequences to the health of the host,as PSA protects animals from experimental colitis (26). Thus, B.fragilis is not only actively inducing its own tolerance, but alsopreserving the integrity of the niche it colonizes for life.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the embodiments of the Tregs, systems and methods of thedisclosure, and are not intended to limit the scope of what theinventors regard as their disclosure. Modifications of theabove-described modes for carrying out the disclosure that are obviousto persons of skill in the art are intended to be within the scope ofthe following claims. All patents and publications mentioned in thespecification are indicative of the levels of skill of those skilled inthe art to which the disclosure pertains. All references cited in thisdisclosure are incorporated by reference to the same extent as if eachreference had been incorporated by reference in its entiretyindividually.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background, Summary, Detailed Description, andExamples is hereby incorporated herein by reference. Further, the hardcopy of the sequence listing submitted herewith and the correspondingcomputer readable form are both incorporated herein by reference intheir entireties.

It is to be understood that the disclosures are not limited toparticular compositions or biological systems, which can, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting. As used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. The term “plurality”includes two or more referents unless the content clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which the disclosure pertains.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the specificexamples of appropriate materials and methods are described herein.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications can bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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1. A method to generate an antigen specific anti-inflammatory regulatoryT cell, the method comprising contacting either: a) a T cell with azwitterionic polysaccharide conjugated to the antigen for a time andunder condition to generate an antigen specific regulatory T cell thatis capable of inhibiting a pro-inflammatory response against theantigen; or b) an antigen presenting cell with a zwitterionicpolysaccharide conjugated to the antigen for a time and under conditionto generate an antigen specific regulatory T cell that is capable ofinhibiting a pro-inflammatory response against the antigen.
 2. Themethod of claim 1, wherein the polysaccharide is PSA.
 3. The method ofclaim 1, wherein the T cell is a regulatory T cell.
 4. The method ofclaim 1, further comprising for b) contacting the antigen presentingcell, after said antigen presenting cell contacting with thezwitterionic polysaccharide conjugated to the antigen, with a regulatoryT cell.
 5. The method of claim 1, wherein for b) contacting an antigenpresenting cell with a zwitterionic polysaccharide conjugated to theantigen is performed in presence of the regulatory T cell.
 6. The methodof claim 1, wherein the zwitterionic polysaccharide is conjugated to theantigen by inclusion of the zwitterionic polysaccharide and the antigenon or within a same vesicle.
 7. The method of claim 6, wherein thevesicle is an OMV.
 8. The method of claim 1, wherein the zwitterionicpolysaccharide is conjugated to the antigen by physical connect.
 9. Themethod of claim 1, wherein the zwitterionic polysaccharide is conjugatedto the antigen by direct or indirect covalent linkage.
 10. The methodclaim 1, wherein the pro-inflammatory response is a cell mediatedinflammatory response.
 11. The method of claim 1, wherein thepro-inflammatory response is a humoral inflammatory response.
 12. Themethod of claim 1, wherein the contacting is performed in vitro.
 13. Themethod of claim 1, wherein the T cells are obtained from an individualpatient.
 14. The method of claim 13, wherein the generated antigenspecific anti-inflammatory regulatory T cells are injected back intosaid individual.
 15. The method of claim 1, wherein the contacting isperformed in vivo.
 16. The method of claim 15, wherein the contacting isperformed by topical or systemic administration of the zwitterionicpolysaccharide conjugated to the antigen to allow generation of theantigen specific T reg in the gut of an individual.
 17. The method ofclaim 1, wherein the antigen is associated with a condition selectedfrom the group consisting of rheumatoid arthritis; myocarditis;scleroderma. type I diabetes, multiple sclerosis, Crohn's disease,ulcerative colitis; Sjorgens syndrome; Hashimoto's thyroiditis; GravesDisease; autoimmune hepatitis; and myasthenia gravis.
 18. A method ofinhibiting antigen specific inflammation in an individual, the methodcomprising treating the individual with a zwitterionic polysaccharideconjugated to a specific antigen for a time and under conditions toinduce an antigen specific anti-inflammatory regulatory T cell in theindividual.
 19. The method of claim 18, wherein the polysaccharide isPSA.
 20. The method of claim 18, wherein the zwitterionic polysaccharideis conjugated to the antigen by inclusion of the zwitterionicpolysaccharide and the antigen on or within a same vesicle.
 21. Themethod of claim 18, wherein the zwitterionic polysaccharide isconjugated to the antigen by physical connect.
 22. The method of claim18, wherein the zwitterionic polysaccharide is conjugated to the antigenby direct or indirect covalent linkage.
 23. The method of claim 18,wherein the antigen is associated with a condition selected from thegroup consisting of rheumatoid arthritis; myocarditis; Scleroderma. TypeI diabetes, multiple sclerosis, Crohn's disease, ulcerative colitis.Sjorgens syndrome, Hashimoto's thyroiditis, Graves Disease, autoimmunehepatitis, and myasthenia gravis.
 24. A method to generate an antigenspecific anti-inflammatory regulatory T cell, the method comprisingcontacting either: a) a T cell with a zwitterionic polysaccharide for atime and under condition to generate an antigen specific regulatory Tcell that is capable of inhibiting a pro-inflammatory response againstthe antigen; or b) an antigen presenting cell with a zwitterionicpolysaccharide for a time and under condition to generate an antigenspecific regulatory T cell that is capable of inhibiting apro-inflammatory response against the antigen.
 25. The method of claim24, wherein the polysaccharide is PSA.
 26. The method of claim 25,wherein the zwitterionic polysaccharide is within or on the cell surfaceof a vesicle.
 27. The method of claim 26, wherein the vesicle is an OMV.28. The method of claim 24, wherein the zwitterionic polysaccharide isconjugated to an antigen.
 29. The method of claim 24, wherein thecontacting is performed in vitro.
 30. The method of claim 24, whereinthe generated an antigen specific regulatory T cells are injected into apatient.